Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon
© da Silva Neto et al. 2010
Received: 26 April 2010
Accepted: 28 August 2010
Published: 28 August 2010
Skip to main content
© da Silva Neto et al. 2010
Received: 26 April 2010
Accepted: 28 August 2010
Published: 28 August 2010
Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat.
In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter.
Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon.
Xylella fastidiosa colonizes the xylem elements of many plants, causing diseases in economically important crops, such as citrus variegated chlorosis in citrus species and Pierce's disease in grapevines . This Gram-negative fastidious bacterium, transmitted by sap-feeding insect vectors, utilizes a plethora of virulence determinants such as adhesins, type IV pili, gum and extracellular cell wall-degrading enzymes to efficiently colonize the plant xylem .
It has been shown that the xylem fluid affects planktonic growth, biofilm formation and aggregation of X. fastidiosa [3, 4]. Xylem is a nutrient-poor environment that contains low concentrations of diverse compounds such as amino acids, organic acids, and inorganic nutrients. Amino acids are the main nitrogen source in xylem fluid of plants, predominantly glutamine and asparagine . Recently, it was determined that glutamine predominates in the xylem sap of grapevine (Vitis vinifera)  while asparagine and glutamine are found in larger quantity in the xylem sap of citrus (Citrus sinensis) . In infected plants, X. fastidiosa grows exclusively in the xylem vessels, where it must cope with nitrogen limitation and be able to utilize amino acids as nitrogen source. Although it has been determined that X. fastidiosa disturbs nitrogen metabolism of infected orange trees , no aspect of the nitrogen metabolism has been investigated in this phytopathogen.
The global response to nitrogen starvation has been studied at the transcriptional level in several bacteria, such as Corynebacterium glutamicum , Synechocystis sp. , Prochlorococcus  and Anabaena sp. . The regulation of nitrogen metabolism is well-established in several model organisms, such as Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum . In E. coli and other enterobacteria, nitrogen limitation causes changes in expression of about 100 genes, whose products are involved in ammonium assimilation and scavenging for nitrogen-containing compounds . Most of these genes are transcribed by the RNA polymerase containing the sigma factor RpoN (σ54) and activated by the nitrogen regulatory protein C (NtrC). The NtrC-RpoN regulon includes at least 14 operons, among them glnAntrBC (glutamine synthetase and the two-component system NtrB-NtrC), glnK-amtB (PII signal transduction protein and ammonium transporter), astCADBE (arginine catabolism), glnHPQ (glutamine transport) and nac (σ70-dependent transcriptional activator) [12, 13]. On the other hand, in the oligotrophic alphaproteobacterium Caulobacter crescentus σ54 does not regulate the majority of genes induced under nitrogen limitation .
Although the most prevalent RpoN-regulated function in bacteria is nitrogen assimilation, this alternative sigma factor controls many distinctive and unrelated cellular functions, such as pili and flagella biosynthesis, plant pathogenicity, catabolism of aromatic compounds and nitrogen fixation . This is possible because σ54 utilizes diverse transcription activators called enhancer-binding proteins (EBPs), all governed by their own signal pathways, for initiation of transcription . Besides the absolute dependence of EBPs and ATP hydrolysis for the formation of the RNA polymerase open complex on the promoters, another unique feature of σ54 is the recognition of -24/-12-type promoters with consensus sequence TGGCACG-N4-TTGC [17, 18]. The σ54 regulon was estimated in several organisms, such as E. coli , Pseudomonas putida  and several species of Rhizobiaceae  by use of powerful computational methods that took advantage of the high conservation of σ54 promoter sequences throughout diverse bacterial groups.
Alternative sigma factors provide effective mechanisms for regulating a large numbers of genes in response to several environmental stresses. In the genome of X. fastidiosa there are genes encoding each of the sigma factors RpoD, RpoH, RpoE and RpoN . Large-scale studies using microarrays and in silico analyses have permitted to determine the RpoH and RpoE regulons and their contribution to the heat shock response [23, 24]. Recently, we have established that RpoN controls cell-cell aggregation and biofilm formation in X. fastidiosa by means of differential regulation of genes involved in type I and type IV fimbrial biogenesis. We have also characterized the first σ54-dependent promoter in X. fastidiosa, controlling expression of the pilA1 gene .
Here, we analyzed the global transcriptional profile of X. fastidiosa under nitrogen starvation conditions using DNA microarrays. A more complete description of the X. fastidiosa σ54 regulon was achieved using microarray data from an rpoN mutant integrated with an in silico analysis of RpoN-binding sites. The regulatory region of the glnA gene that encodes the enzyme glutamine synthetase was further characterized, and confirmed to have a σ54-dependent promoter, suggesting an important role of ammonium assimilation mediated by σ54 in X. fastidiosa.
The citrus strain J1a12 of Xylella fastidiosa  was cultivated in PW medium  without bovine serum albumin and phenol red and supplemented with 0.5% glucose (w/v) (PWG) at 25°C with no agitation. Cultures were also grown in defined XDM2 medium  or XDM2 medium lacking all nitrogen sources (XDM0) at the same conditions. For the rpoN mutant strain , 10 μg ampicillin ml-1 was supplemented to the PWG medium.
For time course studies, late-exponential phase cells in PWG medium were used to inoculate a culture in 100 ml XDM2 medium to an optical density at 600 nm (OD600 nm) of 0.1. Cells were grown during 12 days in the XDM2 medium (mid-log phase) and harvested by centrifugation. Then, the culture was divided into two portions: in one the cells were washed with XDM2 medium, collected by centrifugation and rapidly frozen in dry ice (this aliquot was considered the time zero of the experiment). The second portion was washed with XDM0 medium and the cultivation was continued for 2 h, 8 h and 12 h in XDM0 medium to establish nitrogen starvation conditions. For each time point, cells in a 25-ml culture were collected by centrifugation and rapidly frozen in dry ice, until RNA isolation.
Total RNA was isolated from X. fastidiosa wild type and rpoN mutant cells, grown under nitrogen excess or nitrogen starvation conditions as described above, using the TRIZOL reagent (Invitrogen), according to the manufacturer's instructions. DNA was removed using RQ1 DNase I (Promega). RNA samples were evaluated by electrophoresis on formaldehyde-agarose gels and stored at -80°C. Microarray slides covering more than 94% of all X. fastidiosa genes, spotted at least in duplicate, were prepared as previously described . Fluorescent-labeled cDNA preparation, microarray hybridization, washing and scanning were performed as previously described . The ArrayVision version 6.0 software (Imaging Research, Inc.) was used for spot finding and signal-intensity quantification. Three RNA samples isolated from independently grown cultures of the cells at each starvation period (2 h, 8 h and 12 h) were examined, and each preparation was subjected to microarray analysis. As the genes were spotted at least in duplicate, we obtained six replicates for each gene from three independent data sets per gene per starvation period. Normalization was carried out using the LOWESS algorithm . Differentially expressed genes were identified using intensity-dependent cutoff values based on self-self hybridization experiments . A gene was classified as upregulated or downregulated if at least four of six replicates were outside of the intensity-dependent cutoff curves. Microarray data are available at the NCBI GEO (Gene Expression Omnibus) database http://www.ncbi.nlm.nih.gov/geo, with accession number GSE21647.
Primer extension assays were performed as previously described , using 50 μg of RNA as template isolated from J1a12 or rpoN cells grown in PWG. Total RNA was hybridized to the [γ-32P]ATP-labeled primer XF1842EXT (5'-AACAAAGCGCAAATCGACGAATTCG-3') and extended with the Superscript III reverse transcriptase (Invitrogen). The sequencing ladder was generated with the Thermo Sequenase cycle sequencing kit (USB), using the [γ-32P]ATP-labeled primer M13Forward (5'-GTAAAACGACGGCCAGT -3') and M13 DNA template.
A position weight-matrix was constructed using a set of 186 RpoN-dependent promoters from different bacterial species . This matrix was used to perform a genome-wide screening for putative RpoN-binding sites in the X. fastidiosa genome sequence  with the PATSER module  from the Regulatory Sequence Analysis Tools (RSAT) website . The search for putative RpoN-binding sites was restricted to intergenic regions (non-coding region between two genes) on the coding strand of all annotated genes. Sequence logos were generated using the WebLogo package .
In this work, DNA microarray experiments were used to reveal the global transcriptional profile of X. fastidiosa under nitrogen starvation conditions. The experiments compared changes in the expression profile of cells growing in the absence of nitrogen (XDM0 medium) for 2, 8 and 12 hours compared to cells maintained in defined medium containing amino acids serine, methionine, asparagine and glutamine as nitrogen source (XDM2 medium, zero-time). The relative ratio was calculated for the zero-time sample compared with each time-point sample and data from each point correspond to three independent biological replicates. The complete list of differentially expressed genes is provided in Additional file 1: Table S1 and Additional file 2: Table S2.
Functional classification of differentially expressed genes under nitrogen starvation in X. fastidiosa.
Intermediary metabolism (25/34) #
Central intermediary metabolism (5/10)
Energy metabolism, carbon (3/17)
Regulatory functions (12/4)
Biosynthesis of small molecules (28/25)
Amino acids biosynthesis (13/10)
Nucleotides biosynthesis (2/5)
Sugars and sugar nucleotides biosynthesis (0/1)
Cofactors, prosthetic groups, carriers biosynthesis (8/5)
Fatty acid and phosphatidic acid biosynthesis (4/4)
Polyamines biosynthesis (1/0)
Macromolecule metabolism (28/37)
DNA metabolism (8/8)
RNA metabolism (17/13)
Protein metabolism (3/16)
Cell structure (12/9)
Membrane components (6/3)
Murein sacculus, peptidoglycan (2/0)
Surface polysaccharides, lipopolysaccharides, and antigens (2/1)
Surface structures (2/5)
Cellular processes (9/15)
Cell division (1/3)
Mobile genetic elements (16/7)
Phage-related functions and prophages (8/1)
Plasmid-related functions (7/6)
Transposon- and intron-related functions (1/0)
Pathogenicity, virulence, and adaptation (9/13)
ORFs with undefined category (3/4)
Changes in expression of 20 genes encoding proteins related to transport (8 induced genes and 12 repressed genes) seem to indicate that adjustment of the transport capacity is an important cellular response to nitrogen starvation. There is a predominance of ATP-Binding Cassette (ABC) transporters, possibly involved in the transport of sugars, amino acids and iron, based on sequence annotation (Additional file 1: Table S1 and Additional file 2: Table S2). In E. coli  and Corynebacterium glutamicum  the induction of transport systems of various alternative nitrogen sources is one of the main responses to nitrogen starvation. The repression of genes encoding transporters in X. fastidiosa seems to be an adaptation to long time nitrogen starvation, since most of the 12 downregulated genes were repressed only at the 12 h time point (Table 1 and Additional file 2: Table S2).
In this category, 17 of the 20 differentially expressed genes under nitrogen starvation were repressed, most of them in the 8 h and 12 h periods (Table 1 and Additional file 2: Table S2). Genes of the major pathways of carbon and energy metabolism were repressed, including three genes of glycolysis (pfkA, gapA and fbaB), a gene of the enzyme pyruvate dehydrogenase (aceE), seven genes of the Krebs cycle (acnB, sdhB, lpd, sucB, odhA, sucC and sucD), four genes of the electron-transport chain (etfA, etfB, etf-QO and cyoC) and two genes of the enzyme ATP synthase (atpA and atpD). Downregulation of many genes related to carbon and energy metabolism was also observed when X. fastidiosa cells were exposed to prolonged high temperature  suggesting that this is a common response to long time stress conditions. However, genes for sugar catabolic pathways are induced by nitrogen depletion in the cyanobacterium Synechocystis sp.  and genes encoding glycolytic enzymes and respiratory chain components are upregulated during ammonium limitation in C. glutamicum, maybe due to the necessity of an increased ATP production during nitrogen starvation for ammonium assimilation via the GS/GOGAT pathway .
After two hours of nitrogen starvation, we observed an increase in transcript levels of genes gltD (XF2709) and gltB (XF2710), encoding the two subunits of the enzyme glutamate synthase (GOGAT), while the expression levels of the glnA gene (XF1842), encoding the enzyme glutamine synthetase (GS), was not altered (Additional file 1: Table S1). Assimilation of ammonium by means of the high-affinity GS/GOGAT pathway is more effective than assimilation by the enzyme glutamate dehydrogenase (GDH), under nitrogen limitation. In fact, the genes encoding GS/GOGAT are upregulated under nitrogen limitation in several bacteria [12, 7]. We observed induction of only few genes encoding enzymes involved in catabolism of amino acids or proteins, such as rocF (arginine deaminase), tdcB (threonine dehydratase), pip (proline iminopeptidase) and pepQ (proline dipeptidase) (Additional file 1: Table S1), suggesting that X. fastidiosa might scavenge nitrogen compounds as a secondary mechanism to ameliorate nitrogen starvation. The biosynthesis of amino acids was significantly affected, with 13 genes being induced and 10 genes being repressed (Table 1). However, this may reflect the fact that nitrogen starvation experiments were carried out in XMD2 medium, that contain amino acids (Ser, Met, Asp and Gln). The induced genes encode enzymes that are part of biosynthesis pathways of glutamate, methionine and cysteine, and their induction is probably not related to nitrogen starvation per se, but instead by the removal of these particular amino acids from the medium.
Additionally, the genes encoding RelA and SpoT, two different ppGpp synthetases that produce the nucleotide alarmone ppGpp in response to amino acids or carbon starvation , were induced after 2 h and 8 h of starvation. This upregulation seems to be a sign of intracellular amino acid depletion when X. fastidiosa cells were transferred to XDM0 medium. Increase in the levels of these enzymes might indicate that some functional categories containing differentially expressed genes (RNA metabolism, biosynthesis of amino acids and translation) were affected by the stringent response in addition to nitrogen starvation.
With the exception of the three genes described above (rocF, pip and pepQ), all other differentially expressed genes related to protein metabolism (16 genes) were repressed under nitrogen starvation (Table 1). Among them were genes encoding the major systems of chaperones and proteases of the cell, typical of the heat shock response, such as groEL, groES, hspA, dnaJ, dnaK, grpE, clpB, mopA, htpX, hspA and mucD, and almost all were repressed during the three time-points of nitrogen starvation (Additional file 2: Table S2). These genes are transcribed by σ32 in X. fastidiosa , but the rpoH gene encoding σ32 was two-fold induced in the 8 h and 12 h periods. This strong repression by nitrogen starvation, at least for the groESL operon, could be mediated by the heat-inducible transcriptional repressor HrcA, once the hrcA gene was four-fold induced in 2 h. Severe downregulation in the expression of genes encoding chaperones and proteases of the heat shock response by nitrogen starvation was previously observed in E. coli . Another interesting observation was the differential expression of a large number of genes (23 induced genes and 8 repressed genes) present in the pXF51 plasmid, most of them encoding proteins of the type IV secretion system, involved in bacterial conjugation .
Differentially expressed genes under nitrogen starvation in the rpoN mutant compared to the wild-type strain.
Downregulated genes (positively regulated by RpoN)
5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase
threonine dehydratase catabolic
transcription termination factor Rho
cationic amino acid transporter
Upregulated genes (negatively regulated by RpoN)
recombination protein N
nitrile hydratase activator
conserved hypothetical protein
phage-related repressor protein
conserved hypothetical protein
DNA uptake protein
nitrate ABC transporter ATP-binding protein
NADH-ubiquinone oxidoreductase, NQO14 subunit
Predicted RpoN-binding sites in X. fastidios a genome.
MarR family transcriptional regulator
hypothetical protein (XF0158-59-60)
conserved hypothetical protein (XF0178-79)
hypothetical protein (XF0414-15)
hypothetical protein (probable transposase)
hypothetical protein (XF1315-relA-XF1317-18)
biopolymer transport ExbB protein (exbB-exbD-exbD2-XF0013)
hypothetical protein (XF0507-08)
hypothetical protein (ParB-like nuclease domain) (XF1784-83-82-81)
NADH dehydrogenase subunit A (nuoABCDEFGHIJKLMN)
MFS transporter (XF1749-48-47-46)
30S ribosomal protein S2
preprotein translocase subunit SecE (secE-nusG)
alanyl dipeptidyl peptidase
GTP-binding elongation factor protein
type I restriction-modification system (XF2724-23-22-21)
MFS glucose/galactose transporter (XF1609-10-11)
hypothetical protein (phage-related protein)
hypothetical protein (phage-related protein)
HlyD family secretion protein (XF2093-94)
hypothetical protein (XF2408-09-10)
23S rRNA 5-methyluridine methyltransferase
hypothetical protein (XF0726-27-28-29)
DNA methyltransferase (XF0641-40)
Functional classification of the genes associated to predicted RpoN-binding sites reveals the involvement of σ54 with several cellular functions, such as motility, transcription regulation, transport, carbon metabolism and protein degradation among others. However, a large number of genes (50%) encode proteins that have no attributed function (Table 3). The highest scoring RpoN-regulated promoter was located upstream of the pilA1 gene (XF2542), confirming a promoter previously characterized by primer extension analysis and the role of σ54 in pili biogenesis . The next best hit was found in front of a gene encoding a MarR transcriptional regulator (XF1354), the only regulatory gene associated with RpoN-binding site in our in silico analysis. MarR-like regulators control a variety of biological functions, including resistance to multiple antibiotics, organic solvents, sensing of aromatic compounds and regulation of virulence . A regulatory gene belonging to σ54 regulon could explain how RpoN might indirectly control the expression of genes that are not associated with RpoN-binding sites.
Predicted RpoN-binding sites were identified upstream of four putative operons encoding transport systems: two operons encoding translocases of the major facilitator superfamily (MSF) (XF1749-48-47-46 and XF1609-10-11), one operon encoding resistance-nodulation-cell division (RND) family efflux pump (XF2093-94) and the exbB-exbD-exbD2-XF0013 operon. Genes encoding transporters are regulated by sigma 54 in various bacteria such as E. coli , P. putida  and Rhizobiaceae , although most of these transporters are of the ATP-Binding Cassette (ABC) type. Other functional categories identified were carbon and energy metabolism (nuo operon encoding NADH dehydrogenase and acnA encoding aconitase hydratase), biosynthesis of small molecules (XF1121, XF2677 and XF1315-relA-XF1317-18), DNA metabolism and translation. Possible RpoN-binding sites were also found upstream of two genes encoding putative peptidases (XF0220 and XF2260). In E. coli the ddpXABCDE operon (DdpX is a D-alanyl-D-alanine dipeptidase) is induced under nitrogen limitation, possesses a potential σ54-dependent promoter and seems to work scavenging D-alanyl-D-alanine from peptidoglycan [13, 19]. These results suggest that scavenging of nitrogen compounds could also be a mechanism controlled by σ54 in X. fastidiosa.
To compare microarray data with in silico predictions, the genes and/or operons associated with the 44 predicted σ54-binding sites were cross-examined with the list of genes induced under nitrogen starvation (Additional file 1: Table S1) and the genes with decreased expression levels in the wild type compared to its rpoN derivative mutant (Table 2). Genes encoding the pilin protein of the type IV pili (XF2542) and methylenetetrahydrofolate reductase (XF1121), an enzyme that catalyzes the conversion of methylenetetrahydrofolate to methyltetrahydrofolate, the major methyl donor for conversion of homocysteine to methionine were induced under nitrogen starvation, downregulated in the rpoN mutant and were preceded by σ54-dependent promoters. A set of six genes possessing σ54-dependent promoters (XF0220, XF0308, XF0318, XF0159, XF0567 and XF1316) was induced under nitrogen starvation, but they were not differentially expressed in the rpoN mutant. All other genes showed no consistent correlation between the transcriptome analysis and the computational promoter prediction. These apparent divergences can be attributable to low expression of RpoN- regulated genes unless under specific conditions that activate the enhancer binding proteins, suggesting that both methods are necessary to achieve a more complete description of the X. fastidiosa σ54 regulon. These combined strategies have been applied to determine RpoN regulon in several bacteria, such as Listeria monocytogenes , Geobacter sulfurreducens  and Bradyrhizobium japonicum .
To identify the 5' end of the glnA transcript, primer extension assays were performed with total RNA isolated from the wild-type and rpoN mutant strains. One major cDNA product was observed corresponding to a single transcriptional start site at a cytosine located 35 bp upstream of the glnA re-annotated initiation codon in the wild type strain, but no cDNA product was observed when primer extension experiments were performed with the rpoN mutant (Figure 3B). Upstream of the glnA transcription start site we found the predicted RpoN-binding site, a sequence (TGGTATG-N4-TTGC) that is correctly positioned and matched 9 of 11 nucleotides to the σ54 consensus sequence (TGGCACG-N4-TTGC) (Figure 3C). In other bacteria, glnA has a σ54-dependent promoter and its transcription is regulated by the enhancer-binding protein NtrC . Contact between the activator and the σ54-RNA polymerase complex is achieved by DNA looping, facilitated either by the integration host factor (IHF) protein or by intrinsic DNA topology . In fact, analysis of the regulatory region of the glnA gene revealed the presence of AT-rich sequences with perfect match for the IHF binding site (AATCAA-N4-TTG) besides two putative NtrC-binding sites (Figure 3C).
In conclusion, primer extension data indicate that X. fastidiosa glnA gene has a single canonical σ54-dependent promoter, confirming experimentally the in silico prediction. The fact that sequences related to the NtrC and IHF binding sites exist at appropriate positions upstream of the glnA gene suggested that these factors act in concert with σ54 to initiate glnA transcription. Therefore, ammonium assimilation is a cellular process controlled by σ54 in X. fastidiosa, similarly to that observed in enteric bacteria . Although at high concentrations ammonium is toxic to many plants  and the main source of nitrogen in the xylem sap are amino acids , studies using more precise analytical techniques have detected significant amounts of ammonium in the xylem sap, showing that root-to-shoot ammonium translocation does indeed occur in plants . The ammonium translocated by xylem vessels and that derived from protein catabolism should be used as nitrogen source by X. fastidiosa, through its incorporation into glutamine by glutamine synthetase.
In the present study, we used DNA microarrays to identify global gene expression changes during nitrogen starvation in X. fastidiosa. Nitrogen depletion in XDM2, a defined medium that contains amino acids as nitrogen source similarly to the xylem sap, resulted in major alterations in Xylella transcriptome. Changes in the expression were observed for several genes related to transport, RNA metabolism, biosynthesis of amino acids and translation, as well as a severe downregulation in the expression of genes related to heat shock response and carbon and energy metabolism. However, the function of several genes differentially expressed under nitrogen starvation remains unknown. In addition, we have also obtained a more detailed appreciation of the X. fastidiosa σ54 regulon by combining computational prediction, microarray data and primer extension analysis. Among other cellular processes, RpoN controls pili biogenesis (pilA1) and ammonium assimilation (glnA), consistent with the fact that X. fastidiosa has only two EBPs proteins encoding NtrC and PilR ortologues. Experimental conditions that activate additional genes possessing true RpoN-binding sites remain to be determined.
This work was supported by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). During the course of this work, JFSN and TK were supported by predoctoral fellowships from FAPESP. MVM and SLG are partly supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.