Transcriptomic response of Sinorhizobium meliloti to the predatory attack of Myxococcus xanthus

fmicb-14-1213659 June 13, 2023 Time: 16:41 # 1 TYPE Original Research PUBLISHED 19 June 2023 DOI 10.3389/fmicb.2023.1213659 OPEN ACCESS EDITED BY Christian Sohlenkamp, National Autonomous University of Mexico, Mexico REVIEWED BY David Cole Stevens, University of Mississippi, United States Alessio Mengoni, University of Florence, Italy Christine Kaimer, Ruhr University Bochum, Germany *CORRESPONDENCE Aurelio Moraleda-Muñoz [email protected] †These authors have contributed equally to this work RECEIVED 28 April 2023 ACCEPTED 01 June 2023 PUBLISHED 19 June 2023 CITATION Soto MJ, Pérez J, Muñoz-Dorado J, Contreras-Moreno FJ and Moraleda-Muñoz A (2023) Transcriptomic response of Sinorhizobium meliloti to the predatory attack of Myxococcus xanthus. Front. Microbiol. 14:1213659. doi: 10.3389/fmicb.2023.1213659 COPYRIGHT © 2023 Soto, Pérez, Muñoz-Dorado, Contreras-Moreno and Moraleda-Muñoz. 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Transcriptomic response of Sinorhizobium meliloti to the predatory attack of Myxococcus xanthus María José Soto1†, Juana Pérez2†, José Muñoz-Dorado2, Francisco Javier Contreras-Moreno2and Aurelio Moraleda-Muñoz2* 1Departamento de Biotecnología y Protección Ambiental, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain, 2Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, Granada, Spain Bacterial predation impacts microbial community structures, which can have both positive and negative effects on plant and animal health and on environmental sustainability. Myxococcus xanthus is an epibiotic soil predator with a broad range of prey, including Sinorhizobium meliloti, which establishes nitrogenfixing symbiosis with legumes. During the M. xanthus-S. meliloti interaction, the predator must adapt its transcriptome to kill and lyse the target (predatosome), and the prey must orchestrate a transcriptional response (defensome) to protect itself against the biotic stress caused by the predatory attack. Here, we describe the transcriptional changes taking place in S. meliloti in response to myxobacterial predation. The results indicate that the predator induces massive changes in the prey transcriptome with up-regulation of protein synthesis and secretion, energy generation, and fatty acid (FA) synthesis, while down-regulating genes required for FA degradation and carbohydrate transport and metabolism. The reconstruction of up-regulated pathways suggests that S. meliloti modifies the cell envelop by increasing the production of different surface polysaccharides (SPSs) and membrane lipids. Besides the barrier role of SPSs, additional mechanisms involving the activity of efflux pumps and the peptide uptake transporter BacA, together with the production of H2O2and formaldehyde have been unveiled. Also, the induction of the iron-uptake machinery in both predator and prey reflects a strong competition for this metal. With this research we complete the characterization of the complex transcriptional changes that occur during the M. xanthus-S. meliloti interaction, which can impact the establishment of beneficial symbiosis with legumes. KEYWORDS bacterial predation, myxobacteria, Sinorhizobium meliloti, defensome, bacterial interactions 1. Introduction Bacteria interact with co-habiting microbes in different multispecies communities. The ecological and evolutionary success of microorganisms in a particular environment is not only governed by their capacity to adapt to external abiotic stresses, but also depends on their ability to detect and respond to competition with the neighboring cells. Consequently, Frontiers in Microbiology 01 frontiersin.org fmicb-14-1213659 June 13, 2023 Time: 16:41 # 2 Soto et al. 10.3389/fmicb.2023.1213659 the metabolic processes of one strain are influenced by the metabolic functions of the other members of the community. The relationships between microorganisms range from cooperative symbiotic associations to different competition strategies. In all cases, bacterial interactions involve complex processes that are key determinants that strongly shape the structure of bacterial communities (Bauer et al., 2018;Granato et al., 2019). In the last decades many studies have revealed how bacterial interactions that occur in small communities have consequences that affect in many cases human, animal, and plant health (Pérez et al., 2011; Stubbendieck et al., 2016;Niehaus et al., 2019;Molina-Santiago et al., 2021;Martins et al., 2022). A particular type of interaction is represented by predatory bacteria, which are species that kill and lyse susceptible cells in order to consume the cellular materials as carbon and energy sources (Pérez et al., 2016;Whitworth et al., 2020). Most bacterial predators use two major approaches to kill prey: (i) the endobiotic strategy represented by Bdellovibrio and like organisms (BALOs) that mainly prey on diderm bacteria in the planktonic phase as well as in biofilms (Mookherjee and Jurkevitch, 2022), and (ii) the epibiotic predation exemplified by myxobacteria that can kill and externally lyse a great variety of microorganisms (Muñoz-Dorado et al., 2016;Pérez et al., 2016). The capacity of bacterial predators to kill other bacteria, including multidrug-resistant pathogens, has attracted the attention of researchers as a feasible alternative to antibiotics in the actual crisis (Pérez et al., 2020). As the use of technologies such as next generation sequencing and meta-transcriptomics are increasingly being applied to the study of soil microbiota, bacterial predation is gaining relevant importance as a shaper of microbial communities. Although myxobacteria have been traditionally considered minority components of soil bacterial communities, several studies have revealed that this does not seem to be the case, and that the myxobacterial community is a predominant and highly diverse group within soil niches (Zhou et al., 2014). Until recently, protists have been considered the dominant group preying on bacteria. However, the results of recent studies strongly suggest the importance, and possibly even dominance, of myxobacteria as soil predators. In fact, an analysis of 28 European soils showed that in most of these soils myxobacteria comprise 1.5–9.7% of all obtained SSU rRNA transcripts and more than 60% of all identified potential bacterivores (Petters et al., 2021). Myxococcus xanthus is a soil myxobacterium which has been extensively studied because of its unique complex lifecycle. This lifecycle consists of two stages: a vegetative growth stage in the presence of nutrients and/or prey (it is a facultative predator); and a developmental stage (with the formation of macroscopic fruiting bodies filled of myxospores) when nutrients are depleted (MuñozDorado et al., 2016). It has a large genome which encodes all the genes that participate in the complex social and multicellular lifestyle exhibited during both growth and development (Goldman et al., 2006). Myxococcus xanthus predation requires the participation of many weapons to kill and consume the prey, including a variety of hydrolytic enzymes, outer membrane vesicles, contact-dependent and independent elements, and the production of secondary metabolites such as antibiotics (Pérez et al., 2016;Thiery and Kaimer, 2020;Seef et al., 2021). In addition, this arsenal differs from one prey to another (Thiery et al., 2022). In our laboratory we deciphered the transcriptomic changes that take place in M. xanthus during a complete lifecycle. During development, 1,415 genes were sequentially and differentially expressed in 10 discrete groups (Muñoz-Dorado et al., 2019). Moreover, we have also analyzed the predatosome of this myxobacterium when preying on Sinorhizobium meliloti. The results obtained revealed that the number of genes differentially expressed during predation is lower than during development. Among the transcripts that are up-regulated during predation, the most noteworthy are genes involved in the biosynthesis of secondary metabolites, in the synthesis and degradation of lipids, those encoding both extracellular and outer membrane hydrolytic enzymes, and genes related to social motility and Tadlike apparatuses (Pérez et al., 2022). In predatory interactions, the prey will need to modify the network of genes and pathways required to mount an orchestrated defense against the biotic stress caused by the predatory attack. We will use the term “defensome” to refer to the whole set of genes that vary their transcription in response to the predatory bacterium, either to resist predation or to compete for resources. Nevertheless, some specific defense mechanisms and adaptations of different prey against M. xanthus attack have been reported, so far. For example, galactoglucan and melanin protect S. meliloti from predation by this myxobacterium (Pérez et al., 2014;ContrerasMoreno et al., 2020). The presence of this predator also induces the transcriptional activation of silenced genes coding for antibiotics in Streptomyces coelicolor (Pérez et al., 2011;Lee et al., 2020). Bacillus subtilis induces bacillaene synthesis and forms spore-filled megastructures against the attack of M. xanthus (Müller et al., 2014, 2015). On the other hand, studies of the prey response revealed a novel antibiotic resistance mechanism consisting of the glucosylation of the antibiotic myxovirescin TA, which was discovered in Bacillus licheniformis (Wang et al., 2019). Also, the transcriptome of Escherichia coli against M. xanthus in liquid media has been analyzed, and the results showed that the presence of the predator caused widespread induction in gene expression and enrichment of several pathways including ribosome production, lipopolysaccharide (LPS) biosynthesis, oxidative phosphorylation, production of antibiotics and secondary metabolites, energy and carbon metabolism, and vitamin and amino acid metabolism. However, only the pathway involved in glycerophospholipid metabolism was down-regulated (Livingstone et al., 2018). In the current study, we have analyzed the defensome of the prey S. meliloti against attack by M. xanthus.S. meliloti is an alphaproteobacterium that establishes nitrogen-fixing symbiosis with legumes, thereby contributing to the fertility of soils. This soil bacterium can also exist as a free-living organism in natural environments, where it must adapt to diverse nutrient availability conditions, and compete with other neighboring microbes, including predators such as M. xanthus. These competitive interactions will affect not only the structure of the soils, but also, ultimately, will affect their fertility. Our results reveal that the contact with M. xanthus induces in S. meliloti defense and/or adaptation genes that affect central pathways, such as protein biosynthesis and secretion. The reconstruction of other up-regulated pathways seems to indicate that S. meliloti not only protects itself against predator attack in a passive way by modifying the cell envelope, but also reacts actively by producing H2O2or formaldehyde. The induction of Frontiers in Microbiology 02 frontiersin.org fmicb-14-1213659 June 13, 2023 Time: 16:41 # 3 Soto et al. 10.3389/fmicb.2023.1213659 genes related to iron uptake indicate ion competition. This research completes the study on transcriptomic changes undergone in both partners during the M. xanthus-S. meliloti interaction and draws a panoramic view of the mechanisms and pathways involved in attack, defense, and competition. 2. Materials and methods 2.1. Media, bacterial strains and growth conditions Sinorhizobium meliloti Rm1021 (Meade and Signer, 1977) was used as prey, whereas M. xanthus DK1622 (Kaiser, 1979) was used as predator. Tryptone yeast (TY) solid and liquid media (Beringer, 1974) were used for maintenance and growth of S. meliloti. CTT solid and liquid media (Hodgkin and Kaiser, 1977) were used to grow M. xanthus, and for the predation experiments. Solid media contained 1.5% Bacto-Agar (Difco, Le Pont de Claix, France), and liquid cultures were incubated with vigorous shaking at 30◦C. 2.2. Preparation of prey cells and co-culture of prey and predatory cells Sinorhizobium meliloti was grown in TY broth to an optical density at 600 nm (OD600) of 1 and then diluted using the same broth to a final OD600 of 0.2. Twenty 10-µl drops of the diluted culture were deposited on the surface of CTT agar plates for each replicate and incubated at 30◦C for 24 h. After that time, S. meliloti cells from two replicates were harvested from plates to obtain t= 0 prey samples (samples Sm_t0). Then, to obtain samples of predatory and prey interacting cells, 10-µl drops of M. xanthus, grown in CTT liquid media to an OD600 of 1 and concentrated in TM buffer [10 mM Tris-HCl (pH 7.6), 1 mM MgSO4] to a final OD600 of 15, were deposited on top of each of the rhizobial colonies of a subset of the plates (samples Mx_Sm). Another subset of samples of S. meliloti was kept growing alone (samples Sm). Two replicates from each of the two conditions (predator/prey co-culture and pure culture of S. meliloti) were harvested from plates after 2 and 6 h of incubation. Pellets from each sample were resuspended in 0.5 ml of RNA Protect Bacteria Reagent (Qiagen, Hilden, Germany), incubated at room temperature for 5 min, and centrifuged at 5,000 gfor 10 min (4◦C). Next, pellets were stored at −80◦C. 2.3. RNA extraction To purify RNA, cells were lysed for 10 min at room temperature with lysozyme and proteinase K [250 µl of 3 mg/ml lysozyme (Roche Diagnostic, Mannheim, Germany) and 0.4 mg/ml proteinase K (Ambion, Carlsbad, CA, United States)] prepared in TE buffer [10 mM Tris-HCl; 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0]. RNeasy Mini Kit (Qiagen, Hilden, Germany) was used for RNA extraction [carrying out on-column DNase digestion with the RNAse-free DNase set (Qiagen, Hilden, Germany)], eluting each sample in 50 µl of RNase-free water. 2.4. Library preparation, sequencing, and global transcriptomic data analysis Total RNA samples were processed by Novogene [Novogene Europe, Cambridge, United Kingdom], including rRNA depletion from total RNA samples with the Illumina Ribo-Zero Plus rRNA Reduction Kit (Illumina, Inc.). Remaining RNA was processed according to the procedures described in Pérez et al. (2022). On average, 18.81 million raw reads and a coverage of 421.75x were obtained. After removing reads containing adapters, low quality reads and/or reads with more than 10 percent uncertain nucleotides, the genome coverage varied from 355.53x to 494.52x (median of 415.18x), which provide an excellent coverage of the mRNA fraction. To facilitate comparison analyses with other S. meliloti transcriptomes we used the old nomenclature (SMa_, SMb_ or SMc_), since these identifiers are commonly used in the literature. However, the corresponding new localizers SM_RS are also indicated in the tables. To decipher the defensome, we have considered all the upand down-regulated transcripts with | Log2 Fold Change| >0 and padj <0.05. It must be taken into account that predation is a multifaceted process, and any change could be of interest as we have demonstrated in the case of M. xanthus predatosome (Pérez et al., 2022). The adaptation and defense mechanisms expected may not be very drastic and, for this reason, our research has been focused on those routes in which there is gene enrichment or in those pathways in which many of the genes involved are up or down regulated in the presence of the predator. In all figures and tables the Log2 Fold Change is specified and those Log2 Fold Change >1 or Log2 Fold Change <−1 are highlighted. For researchers interested in this transcriptomics, all data including reads, fragments per kilobase of transcript per million fragments mapped (FPKMs) and Log2 Fold Change are attached as Supplementary material. However, for improved confidence, | Log2 Fold Change| >1 has been used in comparisons with other transcriptomes, and this threshold is also indicated in all the figures and tables. 3. Results and discussion 3.1. Overview of the transcriptomic response of S. meliloti on predatory co-cultures Transcriptional changes in S. meliloti in response to the predatory attack by M. xanthus was investigated by using the RNAseq technology. The M. xanthus-S. meliloti co-culture conditions and the preparation of libraries have been previously described in detail (Pérez et al., 2022). We focused our study on two points in time after contact: at 2 and 6 h, a period of time during which the prey has to adapt its metabolism and structures to the biotic stress that represents the predatory attack. Following the same procedure used to elucidate the M. xanthus predatosome (Pérez et al., 2022), five cDNA libraries were constructed to analyze the S. meliloti defensome: Sm_t0: S. meliloti alone at time 0 h; Sm_t2: S. meliloti alone collected after 2 h on solid CTT medium; Sm_t6: S. meliloti Frontiers in Microbiology 03 frontiersin.org fmicb-14-1213659 June 13, 2023 Time: 16:41 # 4 Soto et al. 10.3389/fmicb.2023.1213659 alone grown for 6 h on solid CTT medium; Mx_Smt2: cells collected after 2 h of interaction between M. xanthus and S. meliloti; and Mx_Smt6: cells harvested after 6 h of the M. xanthus-S. meliloti interaction. From now on, the terms t2 and t6 will be used to refer to results obtained at 2 and 6 h of the co-cultures of M. xanthus and S. meliloti (Mx_Smt2 and Mx_Smt6, respectively) compared to their respective controls (prey cells in pure culture at 2 or 6 h: Sm_t2 and Sm_t6, respectively). The total number of raw reads from each sample, the clean reads (obtained after removal of raw reads containing adapters and/or of low quality reads), the errors Q20 and Q30, and the GC content of the clean reads are compiled in Supplementary Table 1A. The Pearson correlation coefficients (R2) between replicates were satisfactory in all samples (≥0.91) (Supplementary Figure 1A). The principal component analysis (PCA) showed that genes obtained from the same condition cluster together and genes from different nutritional stages and times cluster separately as expected (Supplementary Figure 1B). The RNA mean reads were normalized to FPKM values (Supplementary Table 1B). The FPKM density distributions and the violin diagrams showing similar gene expression levels are depicted in Supplementary Figures 1C, D, respectively. The volcano plots were constructed by using the transcripts of co-cultures at 2 and 6 h versus their respective controls filtered by their fold changes (| Log2 Fold Change| >0) and padj <0.05 (Figures 1A, B). Including novel genes and sRNAs (i.e., non-coding RNAs of 50–500 nt length), 1,772 and 2,081 transcripts were differentially regulated in S. meliloti in response to predator attack at 2 and 6 h, respectively (Figures 1A, B and Supplementary Tables 1C, D). Concerning the direction of transcript regulation, 806 transcripts (45.5%) were up-regulated and 966 (54.5%) down-regulated at 2 h, while 1,108 (53.2%), and 973 (46.8%) transcripts were upand down-regulated, respectively, at 6 h. Overall, these data indicate that 26.5% (2 h) and 31.2% (6 h) of the S. meliloti transcriptome responded to predator attack. This represents a change in transcriptional activity notably greater than that observed in the predator under the same experimental conditions (Pérez et al., 2022). These results are in agreement with those obtained during predation on E. coli, where it was also found that predation caused a much more pronounced response in the prey than in the predator (Livingstone et al., 2018), indicating a strong adaptive, competitive and/or defensive response triggered by the presence of the predator. For further analyses of the defensome in this study, novel genes and sRNAs were not considered, leaving 1,361 and 1,818 transcripts as differentially regulated at 2 and 6 h, respectively. These genes have been organized in 973 up-regulated and 1,139 down-regulated genes identified at 2 h and/or 6 h in Supplementary Tables 2A, B. We investigated whether predation alters gene expression in S. meliloti in a replicon-biased fashion. The S. meliloti Rm1021 genome (6.69 Mb in size) is composed of three large replicons: a chromosome (3.65 Mb) and two megaplasmids, pSymA (1.35 Mb), and pSymB (1.68 Mb) that contain 54, 21, and 25% of the total annotated genes (Galibert et al., 2001). We found that genes differentially regulated during predation were not proportionally distributed among the replicons. Instead, they were biased toward the chromosome (70.6%), whereas only 9.6 and FIGURE 1 Differential gene expression of Sinorhizobium meliloti in response to Myxococcus xanthus predation. Volcano plots of up-regulated and down-regulated genes during the predatory interaction at (A) t2 and (B) t6 (2 and 6 h of contact). The estimated fold changes (x-axis) versus the minus log10 of the adjusted p-values (y-axis) from DESeq analysis are shown in the volcano plots. The significant genes with absolute values of | Log2 Fold Change| >0 and padj <0.05 are depicted in red (up-regulated) or in green (down-regulated). Blue dots indicate non-regulated genes (NO). Gray vertical dotted lines indicate zero-fold change. 19.8% of the differentially expressed genes were associated to pSymA and pSymB, respectively (Supplementary Figure 2A). This chromosomal bias was mainly caused by genes whose expression was increased during predation since 83.2% were located on the chromosome with only 5.6% on pSymA and 11.2% on pSymB. In contrast, down-regulated genes were distributed more evenly, with 59.8% on the chromosome, 13% on pSymA and 27.2% on pSymB (Supplementary Figure 2B and Supplementary Table 3). These results indicate that in response to predator attack, S. meliloti activates chromosomal-encoded functions, whereas the symbiotic plasmids, especially pSymA, have a minor contribution. To identify the main biological processes affected in S. meliloti during predation, two different approaches were used. In one of them, enrichment analyses were carried out using the associated pathways in the KEGG database (Kyoto Encyclopedia of Genes and Genomes; Kanehisa et al., 2021). The up-regulated pathways involved those related to ribosome production, oxidative phosphorylation, and biosynthesis of amino acids, secondary metabolites, and cofactors (Supplementary Figure 3A and Frontiers in Microbiology 04 frontiersin.org fmicb-14-1213659 June 13, 2023 Time: 16:41 # 5 Soto et al. 10.3389/fmicb.2023.1213659 Supplementary Tables 1E, F). The main down-regulated pathways during predation were related to valine, leucine, and isoleucine degradation, microbial metabolism in diverse environments, carbon metabolism, quorum sensing and ABC transporters (Supplementary Figure 3B and Supplementary Tables 1E, F). As a complementary approach, functional categories of the 973 up-regulated (Supplementary Table 2A) and the 1,139 downregulated (Supplementary Table 2B) genes were determined using clusters of orthologous groups (COGs) according to the genome sequence annotations of the S. meliloti Genome Project1(Table 1). Many differentially regulated genes are annotated as either exhibiting partial or global homology to genes deposited in the databases (Not in COGs) or having unknown functions. Nevertheless, several functional categories could be identified associated to up-regulated genes (highlighted in light gray in Table 1), or to down-regulated genes (highlighted in dark gray in Table 1). These analyses suggest that the S. meliloti response to the predatory attack by M. xanthus involves the activation of protein synthesis and secretion, increased energy generation, changes in the cell envelope and membranes, and the stimulation of the transport and metabolism of inorganic ions and nucleotides. At the same time, carbohydrate and lipid transport and metabolism, as well as signal transduction mechanisms and cell division-related functions are repressed during predation. Comparison of the S. meliloti defensome obtained in the present study with that of E. coli (Livingstone et al., 2018) reveals some similarities but also some differences. Thus, increased protein production and energy generation, and the biosynthesis of secondary metabolites were detected during predation on the two preys. However, whereas carbon metabolism and glycerophospholipid metabolism were upand down-regulated, respectively, in E. coli during predation, the opposite regulation was found for the same functional categories in S. meliloti. If these differences reflect different evasion/defense strategies of the prey or if they are the result of different experimental approaches remains unknown. 3.2. Predation on S. meliloti activates protein production and secretion, fatty acid synthesis, and energy generation while repressing fatty acid degradation and carbohydrate transport and metabolism A large fraction (88%) of the genes involved in translation which were identified as differentially regulated in the transcriptome profile exhibited increased expression differentially expressed genes involved in translation identified in our transcriptome exhibited increased expression during predation compared with control conditions (Table 1). Of the 95 up-regulated genes related to translation, 28 code for ribosomal proteins and proteins involved in ribosome maturation and modification, and 33 for aminoacyl-tRNA synthetases and proteins related with tRNA modification. In addition, genes putatively coding for translation initiation (infA,infB,infC) and elongation (tsf,efp) factors, as well 1https://iant.toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi as probable peptide chain release factors (prfA,prfB,prfC) were also up-regulated, strongly suggesting the activation of protein synthesis during predation. Hence, translation is a biological process activated during predation in different prey (Livingstone et al., 2018), but also in the predator (Pérez et al., 2022). In agreement with increased protein synthesis and the consequent greater demand for amino acids, 111 genes involved in amino acid transport and metabolism were also up-regulated. Moreover, genes coding for the ATP-dependent chaperone folding system DnaK/DnaJ/GrpE exhibited increased expression. This system plays a crucial role in microbial proteostasis under both normal and stress conditions by assisting the folding of newly synthesized polypeptides, and of misfolded or aggregated proteins (Winter and Jakob, 2004;Barriot et al., 2020). Recently, S. meliloti DnaJ has been shown to participate in tolerance to different stresses (Brito-Santana et al., 2023). Besides assisting protein folding, the DnaK chaperone also facilitates protein targeting to membranes and protein translocation (Barriot et al., 2020). Interestingly, we found that the majority of genes involved in intracellular trafficking and secretion identified as differentially expressed during predation were also up-regulated (Table 1), suggesting increased protein secretion. This was the case for several genes related to the Sec system, which is responsible for the insertion, translocation, and secretion across the membrane of unfolded polypeptides, which carry a removable N-terminal signal sequence. Thus, genes coding for the membrane-embedded SecYEG translocon, the SecA ATPase and piloting factors such as the signal recognition particle (SRP) Ffh or the SecB chaperone, which maintain newly synthesized proteins in an unfolded conformation and drive them to the membrane (Papanikou et al., 2007), exhibited increased expression during predation. In contrast to the Sec translocon, the Tat system is responsible for exporting previously folded proteins which have a particular signal peptide containing a recognizable twin-arginine motif (Pickering and Oresnik, 2010;Palmer and Berks, 2012). These proteins seem also to be actively exported in S. meliloti during predation, as suggested by the up-regulation of the tatA gene coding for a TatA/E translocase homolog. The up-regulation of protein secretion systems has also been reported in myxobacteria, where it has been associated to the secretion of factors required for predation of bacteria and fungi (Li et al., 2019;Pérez et al., 2022). In the case of the prey, increased protein secretion could be required to maintain bacterial cell envelope protein complexes, whose integrity might be damaged during predatory attack. Many genes coding for the type II fatty acid synthase (FAS II) system, which is responsible for FA synthesis, were found to be up-regulated in S. meliloti during predation by M. xanthus (Figures 2A,2C). These include genes coding for most of the enzymes involved in the initiation phase and elongation cycles of FA chains (López-Lara and Soto, 2018), as well as some paralogous genes. Activation of FA synthesis is also suggested by the increased expression of acpS (smc02654), the holo-acyl-carrier protein (ACP) synthase, which transfers the 4’-phosphopantetheine from coenzyme A (CoA) to apo-ACP, thereby converting it to the functional holo-ACP to which acyl intermediates can be bound. The up-regulation of fabA and fabB required for unsaturated FA synthesis, together with that of fabI and fabF, suggests that the synthesis of both saturated and unsaturated FAs is increased during predation (López-Lara and Soto, 2018). In contrast, FA degradation decreased during predatory attack, as indicated by the Frontiers in Microbiology 05 frontiersin.org fmicb-14-1213659 June 13, 2023 Time: 16:41 # 6 Soto et al. 10.3389/fmicb.2023.1213659 down-regulation of several genes required for this process. Among them, genes coding for proteins involved in the uptake of longchain FAs (fadL), genes involved in the activation of different FAs with CoA (fadD,matB,smc00261,smb20650), as well as genes of the smc02229-fadA-fadB operon, which most likely code for the enzymes required for the different steps in the β-oxidation cycle, are found (Figures 2B,2D). Decreased FA degradation, together with increased FA biosynthesis, could indicate that S. meliloti requires FAs for a function other than energy production, perhaps for building and remodeling the cell membrane in response to predatory attack (see section “3.4. Drastic changes in the S. meliloti cell envelope during predation”). Lipid biosynthesis is also activated TABLE 1 Functional categories of genes differentially expressed in Sinorhizobium meliloti in response to predation by Myxococcus xanthus. Gene category Number of up-regulated genes(1) Number of down-regulated genes Not in COGs(2)123 (43.5%) 160 (56.5%) Function unknown 32 (28.3%) 81 (71.7%) Translation 95 (88%) 13 (12%) Intracellular trafficking and secretion 14 (87.5%) 2 (12.5%) Cell wall/membrane biogenesis 52 (62.7%) 31 (37.3%) Inorganic ion transport and metabolism 55 (61%) 35 (39%) Nucleotide transport and metabolism 36 (61%) 23 (39%) Secondary metabolites biosynthesis, transport, and catabolism 17 (50%) 17 (50%) Transcription 49 (46.7%) 56 (53.3%) Posttranslational modification, protein turnover, chaperones 37 (46.2%) 43 (53.8%) Defense mechanisms 10 (43.5%) 13 (56.5%) General function prediction only 76 (43.2%) 100 (56.8%) Amino acid transport and metabolism 111 (42.7%) 149 (57.3%) Cell motility 11 (42.3%) 15 (57.7%) Replication, recombination, and repair 19 (42.2%) 26 (57.8%) Energy production and conversion 63 (40.9%) 91 (59.1%) Coenzyme transport and metabolism 26 (40%) 39 (60%) Lipid transport and metabolism 33 (37.1%) 56 (62.9%) Signal transduction mechanisms 16 (35.6%) 29 (64.4%) Cell cycle control, mitosis and meiosis 7 (35%) 13 (65%) Carbohydrate transport and metabolism 44 (23.2%) 146 (76.8%) (1)Percent of up-regulated genes within each gene category is shown in parenthesis. Functional categories in which more than 60% of the genes differentially regulated show increased or reduced expression during predator attack are shown in light or dark gray, respectively. (2)COG, cluster of orthologous groups. in M. xanthus during predation, most likely to change the lipid composition of the cell envelope and to synthesize new secondary metabolites that will contribute to kill prey. However, and in contrast to its prey, genes coding for enzymes involved in the βoxidation cycle are up-regulated indicating an increased energy demand during predatory attack (Pérez et al., 2022). Consistent with increased synthesis of proteins and FA, which are metabolically demanding processes, the up-regulation of up to 63 genes involved in energy production and conversion was detected. These include genes coding for different complexes of the respiratory chain and associated functions, such as the chromosomal nuo genes that code for the proton-pumping NADH: ubiquinone oxidoreductase (nuoA1B1C1D1E1F1HIJK1LMN), genes for cytochrome o ubiquinol oxidase (cyoABC), cytochrome c oxidase (ctaCD), cytochrome b (fbcB), and a putative ATP synthase (atpBCF2). Increased expression of genes that code for different enzymes of the tricarboxylic acid cycle was also detected (pdhABC, lpdA1,sdhABCD,fumC,sucA,icd,acnA,mdh). Similar to genes involved in FA degradation, the majority of the genes belonging to the category of carbohydrate transport and metabolism (76.8%) were down-regulated in the transcriptome. Genes coding for different sugar ABC transporters (ugpABC, frcAK,rhaST), glucolytic enzymes (pgm,pgi,pgiA1,cbbA,cbbA2, tpiA1,gap,pgk,pykA), enzymes of the pentose phosphate pathway (cbbT,gnd,eda2,rbsK,ttuD1), and enzymes involved in glycogen (glgC,glgA1) and poly-3-hydroxybutyrate (phbABC) synthesis, showed reduced expression compared with control conditions. Disruption of the glycogen synthase gene glgA1 resulted in decreased polyhydroxybutyrate (PHB) levels and increased EPS levels compared to the wild type (D’Alessio et al., 2017). In starving conditions, the use of glycogen could be an alternative energy source. In favor of this hypothesis is the fact that gene glgX1, involved in glycogen-debranching, is upregulated. 3.3. Increased expression of genes related to iron and phosphorus starvation responses Inorganic transport and metabolism was a functional category that showed enrichment in up-regulated genes identified in the response to predatory attack (Table 1). Within this category, it was remarkable the up-regulation of many genes involved in iron uptake and metabolism, including genes involved in the synthesis (rhbABDEF and sma2339) and uptake (rhtA, rhtX) of the siderophore rhizobactin 1021 (Lynch et al., 2001). Additional genes related with iron and with increased expression are those involved in the uptake of heme and hydroxamate siderophores [shmR,hmuSTV,fhuA2/foxA,fhuA3,fhuP,sma1742 fepG (sma1742), fepB (sma1746)], the exbD gene encoding one of the components of the TonB-ExbB-ExbD complex, which provides the energy for the transport of heme and siderophoremediated iron transport across the outer membrane, the fhuF gene coding for ferrioxamine B reductase, or genes for putative Fe3+ABC transporters [irp6C (smb21429) and irp6B (smb21430)] (Fabiano and O’Brian, 2012;O’Brian, 2015) (Figures 3A–C). Genes coding for regulatory proteins involved in iron homeostasis Frontiers in Microbiology 06 frontiersin.org fmicb-14-1213659 June 13, 2023 Time: 16:41 # 7 Soto et al. 10.3389/fmicb.2023.1213659 FIGURE 2 Changes in fatty acids (FA) metabolism in S. meliloti in co-cultures with Myxococcus xanthus after 2 and 6 h of contact (t2 and t6). (A) Up-regulation of genes involved in FA biosynthesis. ML, membrane lipids (see Figure 4 for more information). (B) Down-regulation of the genes responsible for the β-oxidation degradative pathway. Those genes (up or down-regulated) with demonstrated activity in the literature are indicated by their names (see text for details), while paralogous genes found in the KEGG database and that are also differentially expressed are represented by their gene identifiers. (C,D) Heatmaps of the genes involved in FA biosynthesis and FA β-oxidation, respectively. Red or green edges indicate genes with | Log2 Fold Change| >1, and dotted edges indicate no differentially expressed genes at the indicated time. (Supplementary Figures 4A, B and Supplementary Tables 4A, B) were also differentially expressed during predatory attack. Genes coding for HmuP, which controls hemin acquisition, RhrA, which controls both the synthesis and the uptake of rhizobactin 1021, and the iron regulator Irr were up-regulated, whereas the gene coding for the rhizobial iron regulator A (RirA) was downregulated (Amarelle et al., 2010;O’Brian, 2015). Irr senses iron indirectly through the status of heme biosynthesis (Figure 3D). Under iron-limiting conditions, Irr represses genes encoding proteins that function under iron-sufficient conditions, including rirA (O’Brian, 2015). RirA is an iron-sulfur protein that acts as a repressor of iron-uptake under iron-replete conditions (Chao et al., 2005;Pellicer Martinez et al., 2017;Figure 3B). Therefore, data obtained here indicate that S. meliloti is experiencing iron-limiting conditions during co-culture with the myxobacterial predator, similar to the situation previously reported for S. coelicolor (Lee et al., 2020) and Pseudomonas putida (Akbar and Stevens, 2021). Up-regulation of the iron-uptake machinery was also observed in M. xanthus during co-culture with S. meliloti (Pérez et al., 2022), suggesting that both predator and prey are competing Frontiers in Microbiology 07 frontiersin.org fmicb-14-1213659 June 13, 2023 Time: 16:41 # 8 Soto et al. 10.3389/fmicb.2023.1213659 for iron. It would be interesting to test whether increased rhizobactin 1021 production in S. meliloti Rm1021 enhances rhizobial resistance to myxobacterial predation as shown for P. putida survivors that overproduce the siderophore pyoverdine (Akbar and Stevens, 2021). It is known that the metabolism of iron and manganese are interrelated (O’Brian, 2015). Manganese can replace iron in many enzymes. However, and despite experiencing low iron concentrations, the expression of the mntABCD (formerly sitABCD) genes coding for a manganese ABC transporter (Platero et al., 2003) and that of its regulator Mur were down-regulated during predation (Figure 3E). Several genes related with phosphorous uptake and metabolism were up-regulated in the defensome of S. meliloti, such as those coding for phosphate ABC-type transporters (phoCDE,pstA), phosphonate metabolism (phnGHIJ), alkaline phosphatase (phoX), polyphosphate kinase (ppk), as well as genes coding for regulatory proteins that are crucial for the maintenance of cellular phosphate homeostasis (phoB and phoU). Intriguingly, up to 29 of the upregulated genes in the defensome were also found to be upregulated in the phosphate starvation response of S. meliloti, with the majority of them (25) belonging to the cluster I of PhoBdependent genes induced by phosphate stress (Supplementary Figure 4B and Supplementary Tables 4C, D;Krol and Becker, 2004). PhoB is the response regulator of the PhoR-PhoB twocomponent system that controls a set of genes known as the Pho regulon, which is involved in cell adaptation to phosphate starvation. In addition to genes related to phosphorus uptake and metabolism, the Pho regulon includes other genes such as those related to phosphorus-free membrane lipid biosynthesis (sqdB, btaAB,olsA,olsB), which were also up-regulated during predatory attack. Moreover, 29 genes up-regulated in the defensome increased their expression levels under phosphate starvation in a PhoBindependent manner. This group includes several genes involved in the synthesis of the exopolysaccharide I (EPS I) or succinoglycan (exoA,exoW,exoV,exoK,exoYF1,exoX) (see also Section “3.4. Drastic changes in the S. meliloti cell envelope during predation”). The transcriptional activation of low phosphate-responsive genes, including members of the Pho regulon, could be interpreted as S. meliloti cells facing phosphate-limiting conditions. However, this is difficult to believe considering that the medium used in the experimental setup is a phosphate-rich medium and that phoU is transcriptionally up-regulated. It has been suggested that the regulatory protein PhoU responds to elevated phosphate levels by significantly decreasing the phosphate transport of PstSCAB to prevent phosphate toxicity (diCenzo et al., 2017). Moreover, the activation of EPS I production is known to occur under highphosphate conditions (Mendrygal and González, 2000;AcostaJurado et al., 2021). Therefore, and most likely, the up-regulation of low phosphate-responsive genes in the defensome cannot be the direct result of a phosphorous deficiency. In E. coli, PhoB is not only activated by low phosphate, but also by cell envelope stress (Choudhary et al., 2020). It is not unreasonable to think that compounds and hydrolytic enzymes secreted by M. xanthus during predatory attack disrupt different components of the S. meliloti cell envelope, causing cell envelope stress. Activation of the PhoB regulator by the cell envelope stress caused during predatory attack and/or any other mechanism, together with the known overlap and interaction of the Pho regulon with several other control circuits (Yuan et al., 2006), could explain the differential expression of low phosphate-induced genes during myxobacterial predation. 3.4. Drastic changes in the S. meliloti cell envelope during predation The bacterial cell envelope is a complex structure that provides structural integrity and protects the cytoplasm from changes in the surrounding environment. As a diderm bacterium, the rhizobial cell envelope consists of three layers: the cytoplasmic or inner membrane (IM), a thin peptidoglycan cell wall, and the outer membrane (OM) containing LPS in the outer leaflet. In addition, rhizobia produce different surface polysaccharides (SPSs) that serve different functions during the free-living and symbiotic lifestyles of these bacteria (Acosta-Jurado et al., 2021). Maintenance of cell envelope integrity is essential for viability, and bacteria have developed regulatory mechanisms to defend from envelope disturbance. Considering the epibiotic predatory strategy employed by M. xanthus, it is not surprising that many S. meliloti genes related with the different cell envelope structures are transcriptionally modulated in response to predator attack. Below, we provide interpretation for the transcriptional changes detected in genes associated to different cell envelope structures. In S. meliloti, the exo/exs and wgx (formerly exp) genes are responsible for the production of two different kinds of acidic exopolysaccharides (EPSs): EPS I or succinoglycan, and EPS II or galactoglucan (Becker et al., 2002). The regulation of these EPSs is very complex, and several environmental conditions and regulatory proteins have been involved in their control (Barnett and Long, 2018). As previously mentioned, several exo/exs genes involved in the synthesis of EPS I (exoA,exoB,exoQ,exoF1,exoH,exoK,exoX, exoV,exoY,exoN,exoP,exoW,exoK,exsA,exsI) (Glucksmann et al., 1993) were found to be up-regulated during predation. Concerning EPS II, the S. meliloti strain Rm1021 used in this study lacks a functional ExpR, a LuxR-type regulator that is required for the quorum sensing-dependent production of high amounts of EPS II (Pellock et al., 2002). Nevertheless, the increased expression of wgcA (expC), wgdA (expE1) and that of the wggR (expG) gene that codes for the transcriptional activator of EPS II-related genes, suggest stimulation of EPS II synthesis. Transcriptional activation of EPS II-related genes could be mediated by PhoB, which activates expression of wggR (Bahlawane et al., 2008). As discussed in section “3.3. Increased expression of genes related to iron and phosphorus starvation responses,” the up-regulation of EPS I-related genes is independent of PhoB. In this case, the transcriptional activation of the exo/exs genes could be mediated by regulatory proteins and circuits known to influence EPS I production and whose expression was also induced during predation. This is the case of mucR (Bahlawane et al., 2008), the exoS/chvG gene of the ExoR-ExoSChvI system (Yao et al., 2004;Wells et al., 2007). and the ntrY and ntrX genes of the NtrY-NtrX regulatory system (CalatravaMorales et al., 2017). Recently, the activation of the ChvG/ExoSChvI regulon in response to cell wall stress has been suggested for Alphaproteobacteria (Williams et al., 2022), as well as the coordinated work of the ExoR-ExoS-ChvI and NtrY-NtrX systems to control different functions, including regulation of the bacterial cell envelope (Stein et al., 2021). Frontiers in Microbiology 08 frontiersin.org fmicb-14-1213659 June 13, 2023 Time: 16:41 # 9 Soto et al. 10.3389/fmicb.2023.1213659 FIGURE 3 Iron uptake and rhizobactin 1021 biosynthesis are induced during competition. (A) Sinorhizobium meliloti genes involved in siderophore synthesis and iron-uptake regulation that are induced at t2 or/and t6. Red and blue circles represent Fe3+and Fe2+, respectively. (B,C) Heatmaps of the RirA and Irr dependent genes which are depicted in panel (A).(D) Control of iron homeostasis by the regulators: RirA, RhrA, HmuP and Irr. RirA is a (4Fe–4S) cluster containing protein which represses many genes involved in iron uptake under iron-replete conditions. The manganese responsive Fur-like repressor, Mur, controls manganese uptake. Both global regulatory proteins are down-regulated during predation, indicating a mechanism for the control of iron homeostasis by manganese as has been suggested for other bacteria (see text for details). (Fe–S) clusters are depicted as blue and yellow circles. Brown circles represent Mn2+. Arrows and truncated lines indicate positive and negative regulation, respectively. OM, outer membrane; IM, inner membrane. (E) Down-regulation of rirA,mur and Mur-dependent genes (see text, and Supplementary Tables 4A, B for details). 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