Aggregatibacter actinomycetemcomitans is a periodontal pathogen colonizing the oral cavity of a large proportion of the human population. It is equipped with several potent virulence factors that can cause cell death and induce or evade inflammation. Because of the large genetic diversity within the species, both harmless and highly virulent genotypes of the bacterium have emerged. The oral condition and age, as well as the geographic origin of the individual, influence the risk to be colonized by a virulent genotype of the bacterium. In the present review, the virulence and pathogenicity properties of A. actinomycetemcomitans will be addressed. Aggregatibacter actinomycetemcomitans is a facultative anaerobic Gram-negative bacterium that expresses several virulence factors, which activates a host response that could be associated to the pathogenesis of periodontitis [A. actinomycetemcomitans that contribute to the increased pathogenicity of this species, particularly with regard to early and rapidly progressive forms of periodontal disease [A. actinomycetemcomitans has long been its leukotoxin [A. actinomycetemcomitans is also equipped with a wealth of outer membrane vesicles, like all Gram-negative species, which might confer special virulence properties to this species [A. actinomycetemcomitans within biofilms, characterize the full range of its protein components, and define how these are regulated by other species, and vice versa, when growing within complex polymicrobial communities [dontitis . This re disease ,3, such ukotoxin ,5. Howevukotoxin . Its lipukotoxin . More reukotoxin . A. acti species . There i species . Recent munities . Increasmunities .A. actinomycetemcomitans affects the different leukocyte populations with various death mechanisms [The leukotoxin (LtxA) of chanisms . It actichanisms ,14,15. Ichanisms ,17. In tchanisms ,18,19. Tchanisms   correlates to A. actinomycetemcomitans with enhanced leukotoxicity, including the JP2 genotype and other virulent serotype b isolates [Enhanced leukotoxicity has been reported from serotype b isolates with an intact he toxin . These ihe toxin ,38,42. Aisolates . Interesisolates ,44. In aisolates .The secretion of LtxA is mediated by a Type I secretion system in line with other proteins of the repeat in toxin (RTX family) expressed by several Gram-negative pathogens ,47,48. AThe secreted LtxA has been shown to be efficiently inactivated by proteases and superoxide radicals ,61,62. IA. actinomycetemcomitans has resulted in various genotypes with substantially different virulence properties, i.e., LtxA production [The great genetic diversity of oduction ,28. The oduction . Notablyoduction . ltxA expression, the balance between membrane-associated and secreted toxin, as well as its sensitivity to proteolytic degradation, a gold standard for quantification of LtxA production, is still not available. The first attempt to quantify LtxA activity was to add bacteria to isolated leukocytes and determine cell death by trypan blue exclusion [A. actinomycetemcomitans was higher in young individuals with periodontitis than in older individuals with the disease or in periodontally healthy individuals.Due to the complex regulation of xclusion . In a stxclusion , the leuA. actinomycetemcomitans isolates and support findings from previous leukotoxicity determinations, with enhanced expression in the JP2 genotype [A. actinomycetemcomitans is one of several challenges for future research.Except for examining the leukotoxicity of the isolates, methods targeting gene expression or immunodetection have been developed ,68. Thesgenotype ,70. To oThe CDT family comprises a number of bacterial protein exotoxins that is expressed by several Gram-negative species. Due to its deleterious effects on the host, as revealed in various experimental models, CDTs are likely to be involved in the etiopathogenesis of the associated human infections. They can be described as genotoxins, as their main action is to elicit DNA damage upon the intoxicated host cells . The CDTA. actinomycetemcomitans expresses a CDT and is the only known oral species with this property. An estimated 66% to 86% of its strains express a CDT, and its presence has been associated with the occurrence of periodontal disease [A. actinomycetemcomitans or other microbial constituents of the biofilm community.  disease . It is v disease ,76, ging disease ,78,79,80 disease , as well disease . Cells o disease , B-cells disease , and mon disease . CDT may disease . The delA. actinomycetemcomitans can stimulate the production of several pro-inflammatory cytokines and regulate inflammasome expression irrespective of its CDT, as demonstrated by the use of the CDT-deletion strains [A. actinomycetemcomitans is involved in the etiopathogenesis of periodontitis. On the other side of the bone remodeling equilibrium, when CDT acts directly on pre-osteoclasts, it may also induce apoptosis and hinder their differentiation to osteoclastic cells, thereby contributing a dysbalanced bone remodeling equilibrium that leads to periodontal breakdown [Another potentially pathogenic mechanisms activated by CDT is the stimulation of pro-inflammatory and osteolytic cytokine production by the intoxicated host cells . It has  strains ,88. An i strains . It has  strains ,91, as w strains . This imreakdown .A. actinomycetemcomitans surface is covered by lipopolysaccharide (LPS), a potent pro-inflammatory molecule. A. actinomycetemcomitans LPS comprises a group of structurally related molecules in which the O-specific polysaccharide chain (O-antigen), formed by oligosaccharide repeating units, is the most variable portion , glycero-manno-heptose, glucose, and galactose, and appears conserved among different serotypes [A. actinomycetemcomitans lipid A and core oligosaccharides but in fewer numbers than the commonly found hydroxyl and phosphate group substituents [A. actinomycetemcomitans growth is favoured in slightly alkaline environments [The structures of the core oligosaccharide and the lipid A in erotypes ,97. The erotypes ,97 and perotypes ,101,102.ronments ,104 in wEscherichia coli and Salmonella typhimurium LPS the relative amount of myristic acid is lower than in A. actinomycetemcomitans LPS, while lauric acid and palmitic acid are more frequently found [A. actinomycetemcomitans and Aggregatibacter aphrophilus lipid A [A. actinomycetemcomitans is the presence of both D- and L-glycero-D-manno-heptose, whereas A. aphrophilus LPS only contains the L-enantiomer of this aldoheptose [A. aphrophilus as in LPS from A. actinomycetemcomitans [The fatty acyl composition of lipid A generally varies between different Gram-negative species. For example, in lipid A of ly found . The lip lipid A . Closelyoheptose ,106. By comitans ,105,106.A. actinomycetemcomitans LPS on rodent cells [A. actinomycetemcomitans LPS stimulates human cells. This outlining is done due to the known differences between the murine/rat and human immune systems [A. actinomycetemcomitans LPS are summarized in A. actinomycetemcomitans LPS in junctional epithelium (JE) of a tooth are the epithelial cells. These human epithelial cells have been shown to respond to A. actinomycetemcomitans LPS by expressing IL-15 which results in enhanced IFN-γ production and proliferation of human T cells [A. actinomycetemcomitans LPS causes widening of the intercellular spaces in primary tissue cultures mimicking the JE, a phenomenon not observed with Porphyromonas gingivalis LPS [A. actinomycetemcomitans LPS on the other main human gingival cell type, fibroblasts, have also been reported. Collagen is ingested and digested by gingival fibroblasts in balanced conditions of healthy gingiva. A. actinomycetemcomitans LPS is able to enhance the phagocytosis of collagen by fibroblasts which may result in imbalance in regeneration of the gingival tissue [A. actinomycetemcomitans LPS stimulate the production of IL-6 and IL-8 [Although there are several studies that indicate a distinct effect of nt cells ,110,111, systems . The var T cells . Moreovealis LPS . Variousl tissue . In addiand IL-8 , tissue and IL-8 . The plaA. actinomycetemcomitans LPS has shown to stimulate the expression of microRNA miR-146a [A. actinomycetemcomitans LPS downregulated the expression of miR-32 and miR-29b, which target the pro-apoptotic factor Bim of acute myeloid leukemia cells [A. actinomycetemcomitans LPS has capacity to increase the inducible nitric oxide synthase (iNOS) activity and induce the nitric oxide (NO) production by human osteoblast-like cell line [Gingival tissue contains various resident immune cells, of which macrophages play a central role in periodontitis. miR-146a , which dmiR-146a . On the ia cells  and IL-6ia cells , respectell line . If osteell line .A. actinomycetemcomitans LPS enhances the IL-12 production by mDCs leading to stimulation of IFN-γ expression of natural killer cells and undetectable levels of IL-4, which together may cause the polarization of naïve T cells toward the Th1 type response [A. actinomycetemcomitans LPS originating from different serotypes, serotype b LPS inducing the strongest production of IL-12, IFN-γ, TNF-α, IL-1β, IL-6, and IL-23 [A. actinomycetemcomitans LPS compared to the response to other serotype LPS most likely causes Th1/Th17 type of T cell response in serotype b related infection [Another type of resident immune cells in periodontium is dendritic cells (DCs), which are involved in antigen presenting to T cells. Conventional DCs originate from monocytes, like macrophages, and are called mDCs. Monocytes originating from localized aggressive periodontitis (LagP) patients, recently renamed molar-incisor pattern periodontitis with rapid progression , spontanresponse . There and IL-23 . The difnfection .A. actinomycetemcomitans LPS has been shown to be more potent inducer of neutrophil ROS production than, for example, P. gingivalis or Prevotella intermedia LPS [A. actinomycetemcomitans LPS stimulate the production of inflammatory cytokines IL-1β and TNF-α by PMN more efficiently than P. gingivalis LPS [A. actinomycetemcomitans LPS causes higher production of the above mentioned cytokines as well as IL-6 than E. coli LPS [A. actinomycetemcomitans LPS can shift the movement of monocytes and granulocytes from rolling to the passaging through the vascular wall by downregulating the surface expression of L-selectin and increasing the expression of β2 integrins, respectively [PMNs are innate immune cells which may play both defensive and destructive role in periodontitis . Neutropedia LPS . Moreovealis LPS , and in coli LPS . A. actiectively .A. actinomycetemcomitans LPS, this outer membrane linked polysaccharide has also functions related to bacterial physiology. The serotype specific O-antigen part of A. actinomycetemcomitans LPS takes part in the secretion of leukotoxin, since a serotype b mutant with inactive rmlC and altered O-antigen sugar composition, contained more cytoplasmic and membrane-bound leukotoxin, and secreted less leukotoxin than the wild-type serotype b strain [A. actinomycetemcomitans LPS may mediate direct adhesion to abiotic surfaces [A. actinomycetemcomitans LPS interacts with host molecules, such as human hemoglobin [Despite the vast literature concerning the inflammatory related functions of b strain . Moreovesurfaces . Besidesmoglobin  and IL-8moglobin , which mA. actinomycetemcomitans is one of the human pathogens that is able to bind host cytokines, such as IL-1β, IL-8, and IL-6 [A. actinomyctemcomitans, including intracellular ATP synthase subunit β [A. actinomycetemcomitans. Both Gram-positive Staphylococcus aureus [Pseudomonas aeruginosa [Neisseria meningitidis [and IL-6 ,138, andand IL-6 , which land IL-6 , and chaand IL-6 ,138. Sevubunit β , histoneubunit β , and outubunit β  and secrubunit β . The majs aureus  and Gramruginosa  and Neisngitidis  are ableA. actinomyctemcomitans is able to bind and sense. A. actinomyctemcomitans harbours the two-component system QseCB, which has originally been detected from enterohemorrhagic Escherichia coli EHEC [A. actinomycetemcomitans needs also iron, in addition to catecholamine hormone, to activate the QseC sensor kinase [A. actinomycetemcomitans, and especially the genes needed for anaerobic metabolisms are upregulated [qseC mutant strain forms significantly less biofilm in flow cells than the corresponding A. actinomyctemcomitans wild type and the complemented strains [A. actinomyctemcomitans in vivo. Yet, ΔqseC mutant strain causes less bone loss in murine model of periodontitis than the wild-type strain, suggesting a strong link between A. actinomycetemcomitans QseC and virulence [Human cytokines are not the only host signalling molecules that oli EHEC . In EHEColi EHEC . Howeverr kinase . The Qseegulated . Moreove strains . It is nirulence .A. actinomycetemcomitans OMVs [A. actinomycetemcomitans clinical isolate using Matrix-Assisted Laser Desorption/Ionization Time of Flight-Mass Spectrometry (MALDI-TOF MS) revealed an array of additional tentative virulence-related proteins, including BilRI, Omp100, TdeA, and a ferritin-like protein [A. actinomycetemcomitans OMVs in serum resistance can be hypothesized based on observations that the vesicles could bind to the complement system regulator C4-binding protein in an OmpA-dependent manner [A. actinomycetemcomitans OMVs can transport small molecules such as those contributing to bone resorption, including LPS [A. actinomycetemcomitans OMVs carry NOD1- and NOD2-active peptidoglycan, and upon vesicle internalization into non-phagocytic human cells such as gingival fibroblasts, the OMVs can act as an innate immunity trigger [A. actinomycetemcomitans OMVs contain in addition nucleic acids [A. actinomycetemcomitans OMVs appear to mainly internalize into host cells via clathrin-dependent endocytosis [A. actinomycetemcomitans OMVs, there is no requirement of the toxin receptor LFA1 for vesicle-mediated trafficking of LtxA into host cells [During the latest decades it has become apparent that membrane vesicles (MVs) are naturally shed during growth by many bacteria, archaea, and eukaryotes. Membrane vesicles, also known as “Type Zero secretion”, or referred to as outer membrane vesicles (OMVs) in Gram-negative organisms, serve as a general but relevant mechanism of antigen delivery, and are discharged by both commensal and pathogenic organisms in vivo and when infecting host cells in vitro ,149. Bioans OMVs . OMVs arans OMVs ,152,153. protein ,155. Thit manner . Moreoveding LPS  and lipiding LPS . A. actiic acids . A. actiocytosis  but can ocytosis . Toxins ocytosis , but sucocytosis ,151. Addst cells .A. actinomycetemcomitans is able to grow both supragingivally and subgingivally, corresponding to aerobic and anaerobic growth, respectively. As it can be detected at both locations, it has been postulated that the environment of supragingival plaque harbouring A. actinomycetemcomitans can act as a reservoir for the spread or reinfection of this bacterium of subgingivally [A. actinomycetemcomitans is reported to be detected in the loosely attached unattached plaque area in the middle pocket zone [A. actinomycetemcomitans in periodontal lesions of juvenile periodontitis patients (earlier classification), using culture techniques on disrupted tissue or immunofluorescence microscopy on intact tissue. The former demonstrated the presence of A. actinomycetemcomitans in almost all diseased tissues examined, with evidence of microcolonies and single bacterial cells within the gingival connective tissue, as well as inside the phagocytic cells within the tissue [A. actinomycetemcomitans colony-forming units, which correlated with its presence in the tissue and in the periodontal pocket [A. actinomycetemcomitans in epithelial cells from the lining gingival crevice [A. actinomycetemcomitans is present at a higher prevalence in tissues of younger patients with aggressive periodontitis as compared to chronic periodontitis or health [As a microaerophic organism, ngivally . When grket zone . Earliere tissue . The latl pocket . In situ crevice  or in cl crevice . By quanr health .A. actimomycetemcomitans in biofilms largely comes from in vitro models. In supragingival and subgingival biofilms, A. actinomycetemcomitans does not appear to affect the number of the other species present and appears to form small, dense, and secluded cell clusters of its own species throughout the biofilm mass [A. actinomycetemcomitans exerts regulatory proteomic and metabolic changes in the biofilm, as discussed further.Studies on the localization of ilm mass ,170. YetA. actinomycetemcomitans orchestrates proteomic changes in the context of biofilms. Using 2D gels, Llama-Palacios et al. discovered 87 protein spots differently expressed  between planktonic and mono-species biofilm cultures of A. actinomycetemcomitans [The development of mass spectrometry (MS) technology made it possible to identify multiple proteins in a single run. Therefore, it is a useful tool to study how comitans . Then, ucomitans  despite comitans  as compaA. actinomycetemcomitans within multispecies biofilms is highly relevant. When integrated within a 10-species subgingival biofilm model, A. actinomycetemcomitans did not significantly impact the abundance of the other bacterial species, nor did it affect the biofilm structure, which is consistent with findings in a supragingival biofilm model. Using liquid chromatography–tandem mass spectrometry (LC-MS/MS), 3225 and 3352 microbial proteins were identified in multi-species biofilms in the absence or presence of A. actinomycetemcomitans, respectively [p < 0.05) among these two kinds of biofilms. Interestingly, the regulation trend for individual species was highly individual. For Prevotella intermedia, all quantified proteins were upregulated in the presence of A. actinomycetemcomitans, whereas the majority of the proteins were downregulated for Campylobacter rectus, Streptococcus anginosus, and P. gingivalis. These findings are well in line with the competing growth between P. gingivalis and A. actinomycetemcomitans, shown in a dual-species biofilm [A. actinomycetemcomitans appears to downregulate proteins with ferric iron binding functions and alter the metabolic rate for the overall biofilm.Because oral biofilms occur as complex polymicrobial communities, the study of ectively . Further biofilm . FurtherA. actinomycetemcomitans-containing biofilms on host tissues, the biofilm model was introduced into a bioreactor-supervised 3D cell culture system, which consisted of epithelial and connective tissue structures to mimic the periodontium, as well as monocytes to stimulate the immune response [S. anginosus, A. oris, V. dispar, C. rectus, and P. gingivalis were suppressed when present with the host tissue, while the tissue itself exhibited morphological, immunological, and proteomic changes. The numbers of A. actinomycetemcomitans in the biofilm were not reduced, but more of its proteins were expressed when co-cultured with the 3D (21 proteins) than the biofilm stand-alone (15 proteins) [A. actinomycetemcomitans proteins only comprised a small fraction of all identified proteins in biofilm lysates (21 of 3363) and supernatants (one of 896).To understand the effects of this response . As a reroteins) . Yet, A.A. actinomyctemcomitans virulence properties. We have found that deletion of the gene hns, encoding a histone-like family of DNA-binding, nucleoid-structuring protein (H-NS), a global gene silencer [A. actinomycetemcomitans and decreases its biofilm formation ability when cultured as mono-species biofilms [p < 0.05) were upregulated in the hns mutant biofilm, supporting the role of H-NS as gene suppressor in A. actinomyctemcomitans. Notably, the affected proteins included virulence factors such as leukotoxin A and D (LtxA and LtxD). A similar repression activity of H-NS for virulence factors from other microbes was observed on hemolysin operons hlyCABD [ehxCABD [E. coli and rtxACBD in Vibrio cholerae [hns mutant there was a significant reduction of A. actinomycetemcomitans numbers, without affecting the number of other species. On the protein level, LFQ data suggested that many Fusobacterium nucleatum and Streptococcus oralis proteins were downregulated in biofilm harbouring the wild-type A. actinomyctemcomitans strain, as compared to its hns mutant counterpart, and these proteomic regulations may occur long before the corresponding bacterial growth is affected. Most of the regulated proteins were associated with peptide metabolic process and regulation of translation, supporting a protein-orchestrating role of H-NS in A. actinomycetemcomitans.Attachment to biotic surfaces or other biofilms enhances silencer , leads tbiofilms . LFQ sho hlyCABD  and ehxC[ehxCABD  in E. cocholerae . Within A. actinomycetemcomitans in single-species biofilms and multi-species biofilms is provided in A literature summary of the proteomic findings on A. actinomycetemcomitans strains can be divided into competent and non-competent ones, which refer to their ability to acquire new genes from up-taken extracellular DNA (eDNA) using homologous recombination. Competent strains may have some advantage of being able to uptake eDNA, which could be related to additional means to repair DNA damage, obtain nucleotides and possibly also novel genes [A. actinomycetemcomitans strains are naturally competent [sxy, DNA uptake–related comABCDE, pilABCD, comEAFE1, rec2, and transformation-related comM and urpA [cas system is also closely involved in natural competence, since the loss of CRISPR-cas system is connected to the loss of competence [A. actinomycetemcomitans strains are more prone to HGT caused by plasmids and phages. However, in the non-competent strains, the maintained CRISPRs contains few spacers that most likely are used for chromosomal gene regulation, since they possess specificity for endogenous genes [el genes . Approxiompetent , leadingompetent . It is tompetent . This maompetent . The comand urpA ,188,189.and urpA . In addimpetence . Thus, nus genes .sxy (tfoX), of which levels in the cell is affected by various environmental factors, such as extracellular calcium ions [cas system, the development of competence and expression levels of sxy are stimulated by cyclic AMP [pga gene cluster [pga cluster are needed in the production of most abundant extracellular polysaccharide, N-acetyl-D-glucosamine, in A. actinomycetemcomitans biofilms.The expression on genes of competence locus are regulated by ium ions  and the ium ions . Moreoveclic AMP  and affe cluster , respectA. actinomycetemcomitans strains recognize related eDNA, which is suitable to be taken up, using uptake signal sequence (USS). The Pasteurellaceae family has two types of USS, Hin and ApI, which contain distinctive consensus sequences of nine base pairs. A. actinomycetemcomitans has Hin type USS, consisting of A. actinomycetemcomitans GTGCGGT consensus sequence followed by AT-rich repeats [Naturally competent  repeats ,191. ThiAggregatibacter actinomycetemcomitans is a facultative anaerobic Gram-negative bacterium with the capacity to employ many virulence mechanisms closely associated to the pathogenesis of periodontitis. The variety of virulence properties of this bacterium contribute to the pathogenicity of this species, particularly with regard to early and rapidly progressive forms of periodontal disease. A. actinomycetemcomitans can be found in a large proportion of the human population, and due to the large genetic diversity of this species, several different genotypes or phenotypes with various virulence properties have emerged. Without doubt, individuals carrying A. actinomycetemcomitans genotypes with a proven enhanced leukotoxin production have a significantly increased risk to develop disease. More recent studies have identified suitable genes of this species, which can potentially be traced as markers for epidemiological population monitoring and utilization in individual risk assessment programs. Such bacterial virulence markers in periodontal disease may prove to be important tools in future strategies for personalized dentistry.


Cornus florida and C. kousa , using the chlorotyping system developed on North America-native, wild accessions of C. florida. Our results indicated significant differences in chlorotype frequencies between the two species. Cornus florida samples were represented by all major chlorotypes previously described, whereas all C. kousa samples analyzed had only one of the chlorotype patterns shown by C. florida. The chlorotyping analytic panel was then expanded by sequencing the targeted three non-coding cpDNA regions. Results indicated a major difference in the maternally-inherited cpDNA between the two closely related Big-Bracted Cornus species. Chlorotype diversity and differences in the proportion of informative sites in the cpDNA regions of focus emphasized the importance of proper loci choice for cpDNA-based comparative studies between the closely related dogwood species. Phylogenetic analyses of the retrieved sequences for the other species of Cornus provided information on the relative utility of the cpDNA regions studied and helped delineate the groups  within the genus. Genealogical relationships based on the cpDNA sequences and the inferred chlorotype networks indicated the need for continued analyses across further non-coding cpDNA regions to improve the phylogenetic resolution of dogwoods.Chloroplast DNA is a part of plant non-nuclear genome, and is of particular interest for lineage studies. Moreover, the non-coding regions of cpDNA display higher mutation rates than the conserved coding cpDNA, which has been employed for phylogenetic and population research. We analyzed the cpDNA of 332 gDNA samples from collections of  Cornus L.: Cornaceae) include up to 60 species of shrubs and small trees popular for ornamental and commercial uses , wildlife and human food, and for some ethnic medicinal properties and nine imperfect and compound repeats SSRs were identified (C. florida genome work is completed (currently in progress).As an alternative to the system presented here, several studies used Simple Sequence Repeats (SSR) or microsatellite markers for analyses of cpDNA diversity. In the extreme case, as many as over 200 cpDNA SSR markers were scored. Analyseentified . These eC. florida and C. kousa, and used to infer the levels of genetic diversity and spatial distribution in the native range of C. florida[C. kousa diversity is much less known. Only a few studies analyzed the species in this regard, mostly utilizing the phenotypic data, as part of the Korean conservation efforts[C. kousa wild accessions successfully distinguishing the red- and white-bracted ones, including their origin [The nuclear SSR markers have been developed for . florida, 64–66. n efforts. A smallnd; ref. ). Cultivesistance, 12 or aesistance, 6. In tng system did not s species. In addiion (ITS)–23. ThesC. florida and C. kousa suggests that their diversity has been captured in the cultivated forms at significant levels. Species diversity in the commercial cultivars measured with chlorotyping was lower than in the collection of wild C. florida accessions and suggests the species diversity present in nature will be useful for breeding. Similar comparisons for C. kousa dogwood accessions and cultivars can be completed using the chlorotyping protocols described in this study. We were able to improve on the previous Cornus chlorotyping research by sequencing only five C. florida and six C. kousa accessions over the three non-coding cpDNA regions underscoring the efficiency offered by our approach. Although the C. florida genome sequence is not yet available, our study adds sequence resources to the genus cpDNA information. Furthermore, our data suggests the C. florida-based H3 chlorotype as the most ancestral among the species tested, and underscores the need for testing further regions in regards of their usefulness for Cornus species delineation within the genus and molecular diversity.The presented chlorotyping panel for S1 FigC. florida ; (B) C. kousa ; (C) C. mas ; (D) C. nuttallii . Sequences obtained in the course of this study are appended with “UTK”.Respective Genbank numbers and GUIDANCE (HoT) alignment scores are indicated for the sequences of each species analyzed: (A) (TIF)Click here for additional data file.S2 FigBest Maximum-Likelihood phylogenetic trees of the MAFFT-aligned sequences from cpDNA01 (A), cpDNA02 (B), and cpDNA03 (C) obtained in this study. Sequences were G regions trimmed  after MAFFT alignment to remove the uninformative characters. The RAxML settings of 100 runs and a bootstrap of 10,000 were used. The sequences of Blue-/White-Fruited group served as multiple outgroup. Colored edges indicate the bootstrap support values, with the numerical legends accompanying, respectively. The cpDNA01 alignment was 1667 characters long, and the tree was produced by running 391 alignments, with 3.6% of gaps and completely undetermined characters. The cpDNA02 alignment was 986 characters long, and the tree was produced by running 283 alignments, with 1.6% of gaps and completely undetermined characters. Due to heavy primer looping in the other Cornus species group, seven sequences were omitted Click here for additional data file.S3 FigC. florida , bracts of C. kousa , and leaves of C. florida  were submitted for commercial Illumina HiSeq . Reads produced by the Illumina HiSeq system were subjected to error correction using Rcorrector (RNA-Seq error CORRECTOR)[C. florida or C. kousa transcriptome, respectively[C. controversa  using the BBMap short read aligner with default parameters[Cornus species to the cpDNA; the samtools depth command was used to take this information and determine how many times a query read matched to a reference base[C. florida data, the C. kousa data, and from both species, compared to the C. controversa cpDNA. The results were visualized using R version 3.4.4[C. controversa cpDNA regions . The second tier marks (in light brown) the cumulative coverage of the baseline of NCBI NC_030260.1 and MG525004.1 by the C. kousa transcpritomes (max = 5); base pairs order as in the NC_030260.1. The third tier marks (in magenta) the SNPs and INDELs between C. kousa and C. florida transcriptomes mapped back to C. controversa NC_030260.1 and MG525004.1; no differences were discovered between the two C. kousa cultivars analyzed. The next tier marks (in grey) the coding (high) and non-coding (low) regions of C. controversa NC_030260.1. The next tier marks (in green) the SNPs and INDELs discovered in the transcriptomes of the various C. florida cultivars and accessions analyzed. The next tier marks (in orange) the cumulative coverage of the baseline of C. controversa NC_030260.1 MG525004.1 by the C. florida transcpritomes (max = 6). Finally, the innermost tier marks (in blue) the regions analyzed in detail in this paper, cpDNA 01, 02, and 03. The cpDNA01 and cpDNA 03 partially overlap.Total RNA extracted from the bracts of ORRECTOR). Next, sORRECTOR). Read quORRECTOR). The respectively, using Gpectively. The assarameters. This deence base. Once coion 3.4.4 package ion 3.4.4. The mos(TIF)Click here for additional data file.S1 TableC. florida collection; C. kousa collection; other Cornus species collection), cultivars/lines/accessions were ordered alphabetically. Chlorotyping results as per Call et al.[Within groups Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableCornus species grouping as in ref. [Reported % values  for aligned cpDNA01, 02, 03, and concatenated sequences. Sequenced samples are listed out in (DOCX)Click here for additional data file.S4 Tablehttps://ssr.nwisrl.ars.usda.gov with the default settings.The SSRs were detected in the sequence  using the (DOCX)Click here for additional data file.


Jasmonates are phytohormones that regulate development, metabolism and immunity. Signal transduction is critical to activate jasmonate responses, but the evolution of some key regulators such as jasmonate-ZIM domain (JAZ) repressors is not clear. Here, we identified 1065 JAZ sequence proteins in 66 lower and higher plants and analyzed their evolution by bioinformatics methods. We found that the TIFY and Jas domains are highly conserved along the evolutionary scale. Furthermore, the canonical degron sequence LPIAR(R/K) of the Jas domain is conserved in lower and higher plants. It is noteworthy that degron sequences showed a large number of alternatives from gymnosperms to dicots. In addition, ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs are displayed in all plant lineages from liverworts to angiosperms. However, the cryptic MYC2-interacting domain (CMID) domain appeared in angiosperms for the first time. The phylogenetic analysis performed using the Maximum Likelihood method indicated that JAZ ortholog proteins are grouped according to their similarity and plant lineage. Moreover, ancestral JAZ sequences were constructed by PhyloBot software and showed specific changes in the TIFY and Jas domains during evolution from liverworts to dicots. Finally, we propose a model for the evolution of the ancestral sequences of the main eight JAZ protein subgroups. These findings contribute to the understanding of the JAZ family origin and expansion in land plants. Arabidopsis thaliana and Fragaria vesca [Marchanthia polymorpha, COI1, JAZ co-repressor and MYC TFs are conserved, although the ligand molecule is dinor-oxophytodienoic acid (dnOPDA) [Jasmonates (JAs) are phytohormones that regulate the defense responses, growth and development, fertility and reproduction, as well as the biosynthesis of secondary metabolites in terrestrial plants . Their bia vesca ,4, amongia vesca ,6,7,8. Bia vesca ,11,12,13(dnOPDA) ,15,16. TM. polymorpha it only contains a single JAZ protein [A. thaliana [A. thaliana JAZ1 and JAZ10 proteins [A. thaliana JAZ5, JAZ6, JAZ7 or JAZ8 . For instance, the wheat JAZs TaJAZ4-A, TaJAZ4-B, TaJAZ4-D, TaJAZ5-A, TaJAZ11-A, TaJAZ14-A, TaJAZ14-B and TaJAZ14-D  exhibiteA. thaliana was used as a reference because it is a critical motif for the co-receptor COI1–JAZ complex formation. We took into account that the amino acidic residues at the fifth and sixth position are the most important for the degron functionality [A. trichopoda exhibited LPIARK with higher frequency, as well as VPQARK for gymnosperms  of ionality ,45, thronosperms a. We connosperms a. Finallnosperms a. Thus, nosperms a. Besidenosperms a.A. trichopoda, non-polar amino acids were established at the first four positions and the third position can be occupied by polar residues  at the fifth and sixth positions, respectively. We observed differences depending on plant lineage. For instance, degrons in moss and liverwort maintained the same pattern of biochemical groups at the first four amino acid positions: the first, second and fourth positions corresponded to non-polar amino acids, and the third corresponded to amino acids with polar groups in their lateral chain b. In lycresidues b. In monresidues b. TherefA. thaliana JAZ1, JAZ2, JAZ5 and JAZ6 were grouped together in clade I, which contained ortholog sequences from monocots and dicots  clades . A. thald dicots . Clade Iomestica . Otherwiomestica . In cladomestica . AtJAZ3,omestica . On the omestica . Clade Vomestica . These rhttp://www.phylobot.com/464456268, last accessed date: 19 August 2019). The JAZ protein of M. polymorpha was individually grouped in the first tree branch. Next, the ancestor 518 was common from moss to dicots  by the BLASTP tool. Then, the resulting sequences were filtered. First, partial sequences and those with an e-value > 0.0 were removed. Second, NCBI’s Conserved Domain Database (CDD) was used for the identification of proteins containing the Jas domain, and proteins lacking this sequence were removed. Third, sequences containing PPD or ZML domains were removed. Fourth, redundant sequences were removed after the multiple alignment with those previously reported by using Clustal Omega v2.0.12 . The resulting 1065 JAZ sequences used in the following analysis can be found in JAZ protein sequences of A. trichopoda, monocots and dicots were characterized by multiple alignment with respect to A. thaliana JAZ proteins and by using the Conserved Domains Database (CDD) v3.12 . Other conserved sequences, such as CMID domains and EARs motifs, were searched manually using A. thaliana sequences as a reference. The TIFY and Jas domains of previously reported JAZ proteins were obtained from the information collected in relative publications .The TIFY and Jas domains of newly identified JAZ proteins in moss, lycophytes, gymnosperms, ications . Logo sehttps://www.ncbi.nlm.nih.gov/Class/Structure/aa/aa_explorer.cgi, last accessed date: 12 June 2019).For the degron analysis in the different plant lineages, only amino acidic sequences with Arg/Lys (R/K) at the fifth and sixth amino acidic positions from N- to C-terminus according to the canonical degron sequence LPIAR(R/K)  were conThe evolutionary relationships between JAZ proteins were inferred using 308 full-length amino acidic sequences by using the Maximum Likelihood algorithm and a Jones, Taylor and Thornton (JTT) matrix-based model. The bootstrap consensus trees were inferred from 1000 replicates. Evolutionary analyses were conducted in Molecular Evolutionary Genetics Analysis X (MEGA X)  and from previously reported JAZ sequences. Ancestral sequences were inferred using PhyloBot software  [http://tree.bio.ed.ac.uk/software/figtree/, last accessed date: 16 August 2019). The analysis of JAZ ancestral sequences is available to download at the following URL: http://www.phylobot.com/464456268 (last accessed date: 19 August 2019).For the ancestral JAZ reconstructions, different AtJAZ protein orthologs from different species were used. The amino acidic sequences were obtained from the PLAZA v4.0 database   and usinco, USA)  and MUltco, USA)  with def


CRK genes have been carried out in Arabidopsis and rice, while functional characterization of some CRKs has been carried out in wheat and tomato in addition to Arabidopsis. A comprehensive analysis of the CRK gene family in leguminous crops has not yet been conducted, and our understanding of their roles in symbiosis is rather limited. Here, we report the comprehensive analysis of the PhaseolusCRK gene family, including identification, sequence similarity, phylogeny, chromosomal localization, gene structures, transcript expression profiles, and in silico promoter analysis. Forty-six CRK homologs were identified and phylogenetically clustered into five groups. Expression analysis suggests that PvCRK genes are differentially expressed in both vegetative and reproductive tissues. Further, transcriptomic analysis revealed that shared and unique CRK genes were upregulated during arbuscular mycorrhizal and rhizobial symbiosis. Overall, the systematic analysis of the PvCRK gene family provides valuable information for further studies on the biological roles of CRKs in various Phaseolus tissues during diverse biological processes, including Phaseolus-mycorrhiza/rhizobia symbiosis.Receptor-like kinases (RLKs) are conserved upstream signaling molecules that regulate several biological processes, including plant development and stress adaptation. Cysteine (C)-rich receptor-like kinases (CRKs) are an important class of RLK that play vital roles in disease resistance and cell death in plants. Genome-wide analyses of  Plants encounter many environmental cues during their lifespan. The sessile nature of plants exposes them to a broad range of pathogens, nematodes and symbionts. Plants have developed sophisticated mechanisms to defend themselves against pathogenic and parasitic attacks by evoking their immune responses; however, they establish symbiotic associations with the microorganisms via an equally efficient strategy. The plasma membrane localized receptor-like kinases (RLKs) are the key players in perceiving and transducing these external stimuli to further activate the associated downstream signaling pathways. For instance, RLK elicitation by pathogen/microbe-associated molecular patterns (PAMPs/MAMPs), such as bacterial flagellin or fungal chitin heptamers and octamers, or of host-derived damage-associated molecular patterns (DAMPs), activate the defense pathway. During symbiosis, rhizobia derived lipochito-oligosaccharide (LCO) signals (nodulation Factors) and arbuscular mycorrhizal fungi secreted LCO  and shor8-C-X2-C configuration [CRK genes in rice [CRK members in Arabidopsis [Arabidopsis proteins and in eight Arabidopsis plasmodesmata-located proteins (PDLPs) [Cysteine (C)-rich receptor-like kinases (CRKs), also known as DUF26 RLKs, are a large sub-family of plant RLKs. CRKs have a typical RLK domain structure, i.e., they contain an extracellular domain responsible for signal perception, a single-pass transmembrane domain, and a conserved intracellular serine/threonine (Ser/Thr) protein kinase domain responsible for signal transduction. Most CRKs possess two copies of DUF26 in their extracellular domain. The DUF26 domain contains three conserved cysteine residues in a C-Xguration . The cysguration . The strbidopsis ,7. The D (PDLPs) ,8. Unlik (PDLPs) ,10.Arabidopsis CRK4, CRK6, and CRK36 enhanced the activation of early and late PAMP-triggered immunity (PTI) responses and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv tomato [CRK4, CRK5, CRK13, CRK19, and CRK20 leads to hypersensitive response-associated cell death in transgenic Arabidopsis [CRK7 has been reported to mediate the responses to extracellular reactive oxygen species production [Arabidopsis CRK28 and CRK29 responsible for cell death in association with membrane receptor like protein kinase BAK1 in response to Pseudomonas syringae infection [CRK family members in Glycine max were found to be transcriptionally regulated by the biotic stress signals triggering plant immune response [CRK18 in Gossypium barbadense is reported to confer resistance to verticillium wilt resistance [CRKs are transcriptionally induced in response to abiotic stress conditions such as salicylic acid, ozone, UV light, drought, and salt treatments ,12,13,14bidopsis ,12,15. Coduction . Recent nfection . CRK famresponse . FurtherSymCRK in Medicago truncatula is the only known CRK with a role during symbiosis [CRK genes during legume symbiotic associations.Several RLKs have been implicated in legume symbiosis ,23,24,25ymbiosis ,27. AlthCRK gene family members in Phaseolus vulgaris and compared their expression levels specific to mycorrhizal and rhizobial symbiosis. Further, a detailed analysis of chromosomal localization and phylogenetic relationship among them was carried out. Towards an attempt to understand the phylogenetic distribution of PvCRK family members, gene structural analysis, i.e., intron–exon structure, protein secondary structure, conserved motifs, transmembrane helices, and hydrophobicity were studied. Further, the putative function of each PvCRK was predicted based on the cis-acting elements of the promoters. Expression patterns of PvCRK members in various Phaseolus tissues were studied. Finally, CRK expression patterns from mycorrhiza/Rhizobium inoculated Phaseolus roots are studied using previously generated RNA sequencing (RNA-Seq) data. With these analyses, we provide fundamental data for CRK family genes in P. vulgaris that could be further applied to studying various biological aspects of CRKs in Phaseolus, including symbiosis.In the present work, we identified the Arabidopsis CRK gene family sequences [CRK homologs in P. vulgaris, G. max, M. truncatula, Oryza sativa and Zea mays in the Phytozome genome database (https://phytozome.jgi.doe.gov) using default settings for e-value and the number of hit sequences. The genome versions used for different species were P. vulgaris v2.1, Z. mays PH207 v1.1, M. truncatula Mt4.0v1, O. sativa v7_JGI and G. max Wm82.a2.v1. In addition to Phytozome, various other genome databases were used for the retrieval of CRK homologs, including Legume Information System (https://legumeinfo.org) for legumes, Lotus Base (https://lotus.au.dk) for Lotus japonicus, and OrthoMCL (http://orthomcl.org) for L. japonicus and O. sativa. The respective nucleic acid and peptide sequences were downloaded from the online tool PhytoMine from the plant comparative genomics portal Phytozome v12.1 for further analysis and annotation. Obtained genes and protein sequences were further examined to include the conserved domains by querying using Uniprot [The equences  were use Uniprot  and Pfam Uniprot  databasehttp://meme-suite.org) and signal peptide cleavage site predictor SignalP v4.1 [http://www.cbs.dtu.dk/services/SignalP/). Multiple sequence alignment of intra-species and Phaseolus CRK peptide sequences was performed using ClustalW. Phylogenetic analysis of the aligned sequences was carried out using Molecular Evolutionary Genetics Analysis (MEGA) 7.0 with the neighbor-joining (NJ) method, the JTT+I+G substitution model with 1000 bootstrap replicates keeping the default parameters [http://www.bioinformatics.org/sms2/ident_sim.html).Finally, the putative CRK homologs for each species were filtered using conserved sequence motif analyzer MEME   (http://gsds.cbi.pku.edu.cn/index.php) [http://meme-suite.org/tools/meme). Genes with an e-value of <1 × 10−20 were subjected to further analysis. The motif representation was made with MAST version 4.12.0 ordered by p-values. The motifs obtained were analyzed with the BLASTP interface at National Center for Biotechnology Information (NCBI) website (https://blast.ncbi.nlm.nih.gov/Blast) and Pfam 31.0 database [The exon-intron organization of 46 dex.php) . The condatabase ; each waThe secondary structures of CRK proteins were predicted by MLRC (Multivariate Linear Regression Combination methods) using SOPMA-GOR4-SIMPA and run in NPS@ server  . The outP. vulgaris CRK proteins was achieved by Phobius (http://phobius.sbc.su.se/) [https://web.expasy.org/protscale/) according to Kyte and Doolittle [https://web.expasy.org/protparam/) utilizing the scoring criteria of Gasteiger et al. [Prediction of transmembrane helices of the .su.se/)  server uoolittle . The aver et al. . The grar et al. .http://www.csbio.sjtu.edu.cn/bioinf/plant/) was used to identify the sub-cellular localization of proteins in various cellular organelles and pathways. Plant-mPLoc identifies a wide variety of location sites such as cell membrane, cell wall, chloroplast, cytoplasm, endoplasmic reticulum, extracellular, Golgi apparatus, mitochondrion, nucleus, peroxisome, plastid, and vacuole [Next, Plant-mPLoc tool .The promoter regions 2000 bp sequences upstream of coding region of 46 software  to identhttp://bioinfo.cau.edu.cn/agriGO/) [P. vulgaris. The results are represented graphically.The Gene Ontology (GO) enrichment analysis was performed using AgriGO and REVIGO online tools (agriGO/) . GO catePhaseolus vulgaris L. cv. Negro Jamapa roots colonized with Rhizophagus irregularis spores, or Rhizobium tropici strain CIAT899 [CRK family genes under both types of symbiotic conditions. Heat maps were constructed with fold-change values applying the R package (https://www.r-project.org/). Venn diagrams were drawn with differentially expressed gene (DEG) numbers using a Venn diagram drawing tool (http://bioinformatics.psb.ugent.be/webtools/Venn/).Previously, we performed global transcriptome profiling in  CIAT899 . The preP. vulgaris L. cv. Negro Jamapa seeds and germinated them as described by Nanjareddy et al. [R. irregularis or R. tropici according to Nanjareddy et al. [P. vulgaris plants served as controls. Root tissues were separated from the shoots at two-week post inoculation, and were immediately frozen in liquid nitrogen and stored at −80 °C until they were subjected to RNA extraction. The root tissues were ground in liquid N2, and total RNA was extracted using the Spectrum™ Plant Total RNA Kit  following the manufacturer’s recommendations. DNA contamination in the RNA samples were eliminated by incubating the samples with RNase-free DNase (1 U µL−1) at 37 °C for 15 min and then at 65 °C for 10 min. RNA integrity was verified by electrophoresis, and the concentration was assessed using a NanoDropTM 2000 spectrophotometer . DNA-free RNA samples were used in quantitative real-time PCR assays, which were performed using the iScriptTM One-step RT-PCR Kit with SYBR® Green, following the manufacturer’s recommendations in an iQTM 5 . Each reaction was prepared with 40 ng of RNA as template. A control sample lacked reverse transcriptase (RT) was incorporated to confirm the absence of contaminant DNA. Relative expression values were calculated using the formula 2−ΔCT, where cycle threshold value (ΔCT) is the CT of the gene of interest minus CT of the reference gene. Reference genes viz., EF1α and IDE [To validate the RNA-seq data, we surface-sterilized y et al. . Two-dayy et al. . Uninocu and IDE ,49 were  and IDE . The genCRK genes in P. vulgaris was performed based on homology with the identified Arabidopsis CRK family genes [P. vulgaris CRK homologs from the P. vulgaris v2.1 genome database. A total of 46 CRK gene family members were identified in the genome of P. vulgaris  server. All the 46 CRK members of Phaseolus were found to contain a single transmembrane domain based on the analysis using Phobius, a combined transmembrane topology and signal peptide predictor. The results confirm that the CRK proteins contained only one transmembrane region typical to CRKs and RLKs  and TGA-element (13 genes); gibberellin response dOCT (1 gene), GARE-motif (24 genes) and p-box (7 genes); ethylene and ABA responsive ERE (19 genes) and ABRE (19 genes), were also found on the CRK promoter regions  transcriptome database. Based on the expression profile heat map  values > −1.0) in vegetative  and reproductive tissues . Upregulated  expression in both vegetative and reproductive tissues was seen in an average of 22% CRK genes. However, the change in transcript expression was not detected in an average of 28% CRKs  transcriptome database  was used to classify the 46 CRK genes of function A. MaximuP. vulgaris RNA-seq analysis of mRNA from uninoculated control and 2 weeks post inoculation with mycorrhized or nodulated roots using Ion Proton sequencing; data obtained were then deposited in the NCBI with accession number of PRJNA388751 (https:// www.ncbi.nlm.nih.gov/bioproject/388751). In total, 1959 upregulated and 1260 downregulated mycorrhized DEGs and 1247 upregulated and 1398 downregulated nodulated DEGs were identified [P. vulgaris plants were inoculated with R. irregularis or R. tropici, and total RNA was isolated 2 weeks postinoculation. Later, the transcript abundance of CRK3, CRK12, PvPT4 (P. vulgaris phosphate transporter 4), EnodL12 (early nodulin-like 12) and transcription factor Myb73 was measured by RT-qPCR analysis. The results obtained from both RT-qPCR and RNA-Seq analyses were found to be same  were selected). Out of 46 CRK family members, we observed 24 and 13 differentially responding CRK genes in mycorrhized and nodulated roots, respectively  CRK genes  under mycorrhized conditions and 6 unique (16%) CRK genes under nodulated conditions (4 CRK genes upregulated and 2 downregulated). Seven overlapping (19%) CRK genes were found in both the mycorrhizae and rhizobia colonized roots  of CRK family members either in mycorrhizal or rhizobial symbiosis is poorly understood. Herein, based on mic data  we identectively A, indicaed roots . Among ted roots C. Withinol roots C.PvCRK genes that respond to mycorrhizal and rhizobial colonization. Our observations show that 7, 6, 11, 3, and 3 CRK genes were found in Group I, II, III, IV, and V, respectively  and legumes such as M. truncatula (47 CRKs), G. max (63 CRKs) and L. japonicus (18 CRKs) for the first time.In this manuscript, we identified 46 CRK gene family analysis in Phaseolus shows their distribution in duplicated blocks, implying segmental duplications.The conserved nature of gene structures in the number of introns–exons and motif compositions further indicated fewer insertions and deletions during evolution and supported the theory of gene duplication. While analyzing the gene structures, the exon numbers were similar on intrachromosomal genes rather than on interchromosomal genes. Most CRKs had two DUF26 domains, except for CRK9 and CRK26, which had four DUF26 domains. Evolutionary patterns are attributed to three mechanisms of gene duplications, including segmental duplication, tandem duplication and transposition events such as retro position and replicative transposition . SegmentPhaseolus. In the 3D structure, the hydrophobic cysteine residues will be able to bind to a spectrum of hydrophobic molecules to generate varied cellular responses. Transmembrane proteins contain hydrophobic midsections within the membrane and hydrophilic ends, which are exposed to the aqueous cellular and extracellular environment. Hence, we would hypothesize that the negative GRAVY values are the average of both hydrophilic and hydrophobic residues in the CRK members.Theoretical isoelectric point analysis revealed that the majority of PvCRKs have slightly acidic pH, and the remaining proteins have an alkaline pH. Because plants can vary their gene expression in response to the external pH ,61, the Cis-regulatory element analysis in promoters of CRK members of Phaseolus suggested that they play a role in regulating biotic and abiotic stress responses, and plant growth and development. In the present study, we predicted four types of hormone responsive cis-elements in the promoters of PvCRK genes, including auxin-responsive, jasmonate-responsive, gibberellin-responsive, ethylene-responsive, and salicylic acid-responsive elements. No motifs related to cytokinin response were detected. In Arabidopsis, CRKs are involved in ABA signaling by regulating ABA responses in seed germination, early seedling development and abiotic stress responses. The same Arabidopsis study showed plants overexpressing CRK45 were more sensitive to ABA and hypersensitive to salt and glucose inhibition of seed germination and enhanced drought tolerance, whereas the knockout mutants showed the opposite phenotypes [cis-regulatory elements are another common motif encountered in the promoters of most of the PvCRKs. CRK5 in Arabidopsis is the regulator of UV light responses [Arabidopsis [cis-elements, the presence of fungal elicitor responsive elements signals the putative role of CRKs during mycorrhizal symbiosis. Nevertheless, previous studies demonstrate phytohormones playing crucial roles in defining the rhizobial and mycorrhizal symbiosis. Auxin regulates nodule development by modulating cell divisions and cell cycle genes in L. japonicas and M. truncatula [M. truncatula is known to coordinate nodule organogenesis by integrating bacterial and plant signals [M. truncatula during nodulation [M. truncatula and tomato [enotypes ,53. Lighesponses . Defensebidopsis ,12,15. Auncatula ,66. The  signals ,68. Furtdulation . Althougdulation ,71. Gibbdulation ,75,76,77dulation ,79,80. Ad tomato ,82,83,84d tomato ,86,87.PvCRK genes show differential expression patterns in various vegetative and reproductive tissues of Phaseolus. RT-qPCR analysis of representative CRK genes  in different Phaseolus organs show variable expression patterns and were consistent with the Phytozome transcriptome database. As shown in the results, out of 46 PvCRKs, an average of 50% of genes showed low expression and 22% of genes exhibited high expression in all tissues analyzed. Curiously, CRKs with high expression in most of the tissues belonged to phylogenetic group III, implying their indispensable role in various aspects of growth, development, reproduction and defense in homologues in other plant systems [ systems ,14,15,16Rhizobium/mycorrhiza inoculated Phaseolus for CRK gene expression, interesting facts were uncovered. With the applied cutoff value of the transcripts, 24 CRK genes were upregulated and none were specifically downregulated. However, under Rhizobium inoculated conditions, 4 CRK genes were downregulated and 9 were upregulated. Seven CRKs were shared between the symbiotic conditions. Taken together, 11 CRKs with high expression under mycorrhiza/Rhizobium inoculated conditions belonged to group III of the phylogenetic tree of Phaseolus CRKs.While analyzing the previously reported  RNA-Seq CRK family of P. vulgaris and identified 46 PvCRK genes. An array of the biochemical characteristics of the PvCRK proteins was analyzed. The phylogeny of the PvCRK members classified them into five groups, which were substantiated with the similarities found in the gene structure and motif arrangements. PvCRK genes were distributed on 8 chromosomes among 11 chromosomes of Phaseolus. Gene clustering was the most common feature of the CRK gene distribution, and the largest cluster of 22 genes (CRK16 to CRK37) was found on chr07. Gene clustering indicated the possibility of gene duplication as a factor of CRK gene family expansion. PvCRK members were differentially expressed in all vegetative and reproductive tissues of P. vulgaris. Further, GO analysis revealed divergent roles of CRK proteins in Phaseolus. RNA-Seq data for mycorrhiza/Rhizobium colonized Phaseolus root tissues revealed shared and unique PvCRK genes that could play a decisive role during symbiotic events. This article also provides a comprehensive list of CRKs in L. japonicus, M. truncatula and G. max and Z. mays as a first report. Taken together, the results provide a foundation for functional characterization of CRK proteins in Phaseolus and also other species discussed.In summary, we performed a genome-wide analysis of the


Leishmania major, which may be crucial during cytodifferentiation and the environment adaptive responses of the parasite. Leucyl, phenylalanyl-tRNA protein transferase and arginyl-tRNA protein transferase are key components of this pathway in E. coli and eukaryotes, respectively. They catalyze the specific conjugation of leucine, phenylalanine or arginine to proteins containing exposed N-terminal amino acid residues, which are recognized by the machinery for the targeted proteolysis. Here, we characterized a conserved hypothetical protein coded by the LmjF.21.0725 gene in L. major. In silico analysis suggests that the LmjF.21.0725 protein is highly conserved among species of Leishmania and might belong to the Acyl CoA-N-acyltransferases (NAT) superfamily of proteins. Immunofluorescence cell imaging indicates that the cytosolic localization of the studied protein and the endogenous levels of the protein in promastigotes are barely detectable by western blotting assay. The knockout of the two alleles of LmjF.21.0725 by homologous recombination was only possible in the heterozygous transfectant expressing LmjF.21.0725 as a transgene from a plasmid. Moreover, the kinetics of loss of the plasmid in the absence of drug pressure suggests that maintenance of the gene is essential for promastigote survival. Here, evidence is provided that this putative aminoacyl tRNA-protein transferase is essential for parasite survival. The enzyme activity and corresponding post-translational regulatory pathway are yet to be investigated.The N-end rule pathway leads to regulated proteolysis as an adaptive response to external stress and is ubiquitous from bacteria to mammals. In this study, we investigated a gene coding for a putative core enzyme of this post-translational regulatory pathway in  Leishmania. The disease is highly prevalent, with 12 million cases distributed in more than 80 countries and an estimated population of 350 million at risk of infection. Two million new cases are reported annually , was used as a control line.Individual clones of +/PAC/(+) and conditional null mutant, HYG/PAC/ (+) were isolated from the M199 solid medium (2% agar plates). The media were supplemented with each drug or a combination of puromycin, hygromycin B and G418 in concentrations corresponding to 4 x IC7 promastigotes) were washed twice in PBS/FBS (1%). An equal number of L. major (WT) parasites were used as a negative control, and 100,000 events were recorded for both the L. major transfectant and wild-type parasites. Promastigotes from all transfectants were harvested (3x107 cells) after ten passages in the absence of drug pressure.GFP expression level was monitored using a FACSCalibur flow cytometer (BD Biosciences). Parasites (2x10L. major WT and +/PAC/(+) transfectant  were pelleted and washed twice in PBS. Poly-lysine coated slides (0.01%) were prepared, and parasites were diluted to 1:10 and microscopically examined. The parasites were applied to the slides after fixation in 3% formaldehyde; non-adherent cells were removed by a gentle wash with PBS. The slides were blocked with 2% BSA, 0.1% Triton X-100 in PBS for 1 hour, followed by incubation with anti-LmjF.21.0725 (1:500 in blocking solution) for 1 hour. The cells were washed three times with PBS and then incubated with F (ab') 2-Goat anti-Rabbit IgG (H+L) Secondary Antibody conjugated with Alexa Fluor (1: 400 in blocking solution) for ½ hour. The cells were washed three times with PBS, and a sufficient quantity of DAPI was applied. After washing the slides three times, coverslips were applied, and the slides were left for 24 h, at 4°C.For the subcellular localization of LmjF.21.0725, promastigotes of LmjF.21.0725, we performed southern blotting analysis. Briefly, we used 10 μg of genomic DNA from the following L. major lines: wild type/parental (+/+); heterozygous (+/PAC); heterozygous with the LmjF.21.0725 expressing vector (+/PAC/(+)) and a conditional transfectant HYG/PAC/(+) digested with SalI and fractionated in 0.8% agarose gel. Blotted nylon membranes were probed with the DNA fragments internal to the coding sequences for hygromycin phosphotransferase , puromycin acetyl transferase  or LmjF.21.0725 (472 bps). Hybridization and washing steps were performed following the manufacturer’s instructions .To analyze the correct recombination events and to check for the presence/absence of endogenous Genomic DNA from the studied strains was embedded in low-melting-point agarose blocks treated with proteinase K, as previously described . ElectroLmjF.21.0725 cassette into the ribosomal SSU locus of the genome of L. tarentolae, a non-pathogenic host useful for overexpression of Leishmania proteins [LmjF.21.0725 was cloned between BglII and NheI  of prechilled L. tarentolae cultured cells were transfected. Electroporation was performed in a pre-chilled 2-mm cuvette  using a time range of 5.5–5.6 ms/pulse. The electroporated cells were further incubated on ice for 10 minutes and then transferred to 10 ml of BHI medium for 20 hours. The cells were gently dispersed on BHI agar plates supplemented with G418 (50 μg/ml). After ten days of incubation at 26°C in the dark, colonies (1–2 mm diameter) were picked and cultured in 1 ml of LEXSY selective media for 2–3 days. The clones were microscopically examined for growth, motility, and shape. To screen for LmjF.21.0725 expression, 500 μl of the densely grown culture were analyzed by Western blotting using specific anti-LmjF.21.0725 and anti-histidine antibodies. The positive clones were then transferred to 10 ml of BHI selective medium and grown to OD 1.4 .The pLEXSY_NEO2 expression vector  was employed for intracellular expression of LmjF21.0725. The construct was designed for the targeted integration of the proteins . The CDSPurification of the recombinant protein was performed by immobilized metal-affinity chromatography (IMAC) on 1 ml of HIS-Select® Nickel Affinity Gel (Sigma) following the manufacturer's instructions. Cell lysis was performed with a phosphate buffer solution containing 1% NP40, pH 7.4. Elution was performed with a gradient of imidazole concentration from 20 to 500 mM. The purified rLmjF.21.0725-HIS eluted at approximately 320 mM of imidazole. For the MS analysis, the protein was fractionated on 12% polyacrylamide gel  and stained with MS-compatible colloidal Coomassie staining . The gelLmjF.21.0725 is annotated as a gene coding for a conserved hypothetical protein of unknown function. A sequence similarity search using BLASTp against the non-redundant protein database (nr) of NCBI resulted in 83%-100% sequence identity with conserved hypothetical protein sequences of various species of Leishmania from both subgenera  , and Phytophthora nicotianae   . Interese 2e-10) .rd and 322nd amino acid positions. In addition, using the PANTHER classification tool, the Lmj.21.0725 predicted product was also identified as a putative L/F transferase  (α backbone root-mean-square deviation (RMSD) and TMscore were 2.575 Å and 0.82431, respectively. Considering the higher possibility of the active sites location on the large pocket of the protein structure, we determined the largest pocket on the predicted structure. Use of the suite “Phyre2 investigator” further confirmed the high quality of the predicted Lmj.21.0725 structure   superfamily domain between the 133nsferase . We prednsferase . The sofse, PDB) . Phyre2 se, PDB) . The dettructure . Overallity-17%) .B. saltans, a free-living flagellate protozoan, and classified kinetoplastids, oomycetes, aconoidasida and conoidasida as the four separated taxons. Interestingly, the inferred tree also discriminated the Leishmania species into the subgenera Leishmania and Viannia as two distinct monophyletic lines, indicating the Lmj.21.0725 sequence as a potential phylogenetic marker for Leishmania spp.  with a catalytic activity of an arginyl-tRNA protein transferase, which is typical in eukaryotes. LmjF.21.0725 showed limited homology with the P. falciparum enzyme   . We condauritius .L. major genome using linearized KO cassettes . Another round of transfection to delete the second allele encoding LmjF.21.0725 was then successful, and several independent clones called PAC/HYG/(+) were obtained.To understand the function of LmjF.21.0725, we planned to delete the two alleles  from theassettes . HoweverLmjF.21.0725 locus, indicated by a 7.5–8 kb band instead of the predicted 4.7-kb band , in addition to the LmjF.21.0725-plasmid-derived (pXNeoGFP-LmjF21.0725) bands  transfectant, conducted by southern blotting, suggested that an extra copy of the endogenous gene was present. When using the HYG probe, we confirmed the proper integration of only one allele in the correct locus, and the predicted 5.3-kb band was observed . Neverth-kb band . This banctional , althougL. major strains. The results with these five independent PAC/HYG/(+) transfectants were also suggestive of the presence of an additional endogenous copy of LmjF.21.0725 and an abnormal additional fragment probing for endogenous LmjF.21.0725 and PAC marker, respectively (We repeated the southern blot experiment with five randomly selected PAC/HYG/ (+) clones, the heterozygous clone +/PAC and two wild-type ectively . The extectively . HoweverLmjF.21.0725, the above-described PAC/HYG/ (+) lines were used to evaluate the plasmid maintenance under no drug pressure. We conducted a comparative analysis of the kinetics of plasmid loss on the control cells and PAC/HYG/ (+) transfectants as a measure of the relevance of the plasmid/exogenous gene for the survival of the parasite. The parasites were kept in culture in the absence of drug for ten serial passages and submitted to FACS analysis to examine the dynamics of plasmid loss by monitoring GFP expression levels. We observed that, differently from the control lines, all four tested PAC/HYG/ (+) independent transfectant clones maintained the plasmid bearing exogenous LmjF.21.0725 and GFP (>76% of cells in each culture retained the plasmid). In contrast, the dynamics of plasmid loss of the heterozygous mutants (+/PAC(+)) were fast, and only ~17% of the cells maintained the plasmid after 10 serial passages, as indicated by the distribution of cells with an intense fluorescent signal  and in the LmjF.21.0725 transfectants evaluated . A shared limited sequence homology between L/F transferase of E. coli and LmjF21.0725, makes it difficult to predict the active site, however a large hydrophobic pocket was detected in our predicted model, that might be the active site , a close relative of the nonribosomal peptidyltransferase . The enzE. coli . The N-eE. coli . Althougy others , 45, no ive site . We mustP. falciparum has been characterized with a structure similar to the bacterial L/F transferase but with the catalytic activity of an arginyl-tRNA protein transferase [P. falciparum and LmjF.21.0725 . Interestingly, the sequence conservation was high among the homologues of LmjF.21.0725 from various species of Leishmania, different from the higher divergence noted among putative L/F transferases from various Plasmodium species. We suggest that LmjF.21.0725 might be a phylogenetic marker capable of differentiating the protist parasite into respective classes and Leishmania species into the subgenus Leishmania and Viannia, as two distinct monophyletic lines. Nevertheless, the 3D model and primary sequence analyses show reasonable similarity/identity to the E. coli sequence and could be a limiting factor to differentiate species. This evidence is preliminary and further investigation must be conducted to confirm the hypothesis.Interestingly, a protein homologue from nsferase . Our anaLmj.F21.0725 product, we first attempted to disrupt both alleles. We obtained the heterozygous line but did not succeed in generating a double knockout. Hypothetically, this result indicated the possible essentiality of the gene for the promastigotes of L. major. This hypothesis was reinforced after obtaining the double replacement of two alleles with selectable markers in the strain transfected with the plasmid expressing LmjF.21.0725 [in vitro proliferation. Moreover, to confirm the presence and distribution of the hypothetical protein encoded by LmjF.21.0725, we exploited an anti-peptide antibody against the predicted amino acid chain of the coded protein. Immunofluorescence analysis confirmed the presence of a very low level of the expected protein in the wild-type L. major promastigotes. These results corroborate a previous report showing very low levels of the expression of endogenous LFTR in E. coli [E. coli and S. cerevisiae; only the L. tarentolae expression system gave satisfactory results. Nevertheless, the amount of protein obtained was too low to establish the biochemical assay necessary to confirm its enzymatic activity. The purified protein was confirmed by Western blot analysis. The SDS-PAGE-purified rLmjF.21.0725 was further subjected to mass spectrophotometry, which confirmed its identity and validated it as a product of the studied protein encoding ORF , which would be only explained by the lack of cleavage at the referred restriction site. The conditional null mutant was shown to have an additional copy of endogenous LmjF.21.0725; these results were consistently observed with 4 independent clones. A BLAST-n search using the LmjF.21.0725 sequence against the L. major genome deposited at the TriTrypDB showed no additional copies of the gene. We further investigated possible ploidy or somy changes using pulsed field gel electrophoresis, but no differences were observed in the different lines, indicating no ploidy or full-chromosome copy number modifications. Our investigation does not rule out smaller regions of altered chromosomal structure, as it is known that events of partial somy or complete ploidy changes while inactivating an essential gene have been reported for Leishmania [L. infantum (GSH1); in this case, a single allele replacement resulted in the emergence of an extra copy of the gene [Leishmania donovani is associated with the adaptability of the parasite [Analysis of the wild type and transfectant ishmania , 49–52. the gene . Furtherparasite .LmjF.21.0725 in the promastigotes of L. major was also assessed via analysis of the dynamics of passive loss of the plasmid in the absence of the drug of selection, after serial passages using five independent conditional null mutants. The results corroborate previous results that suggested that the presence of LmjF.21.0725 is essential for the survival of L. major promastigotes.The essentiality of Leishmania parasite are targeted for the discovery of new drugs [Leishmania major genome has been sequenced, assembled and thoroughly annotated, representing a relevant resource for exploration of drug target candidates. Nevertheless, many genes do not yet have a predicted function and are assigned as hypothetical proteins encoding genes. Among them, several potential drug targets have been identified using in silico approaches [P. falciparum, which has also shown expanded substrate specificity and is suggested as a novel target for anti-malarial drugs [Leishmania-specific ubiquitin-related modifier (Ufm1) protein, which was shown to be functional and involved in the pathogenesis of L. donovani, is essential for cell division at the amastigote stage [LmjF.21.0725 homologue has not yet been characterized from any species of Leishmania, and the available literature falls short of revealing any potential role in the aminoacyl-tRNA-protein transferase pathway in Leishmania cytodifferentiation and adaptation within the host macrophage. This study is the first in silico analysis to suggest the presence of a conserved putative aminoacyl-tRNA-protein transferase in Leishmania. Thus far, no attempts have been made to determine natural substrates or other biochemical properties of this enzyme. Experimental investigation is required to establish the catalytic activity of the LmjF.21.0725 protein in Leishmania and its putative role in virulence and pathogenesis.Essential pathways in parasites are key to the validation of new drug leads ; 56, andew drugs –59. Despproaches –63. Inteal drugs –65. Regual drugs . Leishmate stage ,68. To tLeishmania species, and more importantly it is essential for parasite survival as a promastigote. Therefore, it is relevant to further investigate if this protein is also essential for amastigotes survival in vivo.In conclusion, the current report is a stepping stone for future research. The hypothetical protein LmjF.21.0725 is a structural homologue of N-acyl aminotransferase (NAT) superfamily proteins, it is highly conserved among the S1 FigL. major. (B) & (C) Vectors used for homologous recombination at Lmj21.0725 locus to replace endogenous gene with selectable markers: Hygromycin Phosphotransferase  and Puromycin Acetyl Transferase , respectively. (D) and (E) Targeting fragments obtained by restriction digestion of depicted vectors with ApaI/StuI  and NotI digestion , respectively.(A) Schematic diagram showing strategy of LmjF21.0725 in (TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(A) Overall antigenicity of the LmjF.21.0725 indicating region selected for antigenic peptides ; (B) Hydrophobicity plot of the LmjF.21.0725 sequence showing the amino-acid sequence location of used peptide- (Peptide 4 = LFTR4- used for the study).(TIF)Click here for additional data file.S4 Fig(A)In silico pLEXSY-NEO-Lmj21.0725 circular construct. (B) Agarose gel showing SwaI digested fragment of linear cassette for integration in SSU locus (6 kb). (C) Western blot analysis of whole-cell lysate of independent transfectant confirming expression by Western blotting using anti-HIS antibody (D) Western blot of fractions eluted with 500 mM imidazole using anti-Lmj21.0725 antibody; W- wash, FT- flow-through lysate, WCL-whole-cell lysate of L. tarentolae transfectant. (E) Polyacrylamide gel (12%) showing the purified Lmj21.0725 (42 kDa) (F) Confirmation of rLmj21.0725 using trypsin-digested mass spec analysis showing alignment between different identified peptides and deduced amino-acid sequences of LmjF.21.0725.(TIF)Click here for additional data file.S5 FigA) Hydrophobic surface (Red color) of the predicted model of LmjF21.0725 predicted by PyMOL. (B) Largest predicted pocket of LmjF21.0725 indicates the possible active site determined by Phyre2 server using the Phyre2 investigator suite (C) LmjF21.0725 sequence showing the amino acid residues involved in formation of the pocket (Red color).((TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.


Mycobacterium tuberculosis and this bacterium is particularly successful at surviving and replicating within macrophages. Here, we will highlight the metabolic processes that M. tuberculosis employs during infection in macrophages and compare these findings with what is understood for other pathogens in the M. tuberculosis complex.Pathogenic mycobacteria are known for their ability to maintain persistent infections in various mammals. The canonical pathogen in this genus is  Mycobacterium tuberculosis is the causative agent of human tuberculosis (TB) and this bacterium is a member of the closely related cluster of species termed the Mycobacterium tuberculosis complex (MTBC). Members of the MTBC cause TB or TB-like disease in humans, livestock, and wildlife with high levels of morbidity and mortality. It is thought that all MTBC pathogens evolved from a common ancestor yet the bacteria fall into phylogenetically distinct lineages and clades under the following species names: M. tuberculosis, M. africanum, M. microti, M. bovis, M. suricattae, M. mungi, M. orygis, M. caprae, M. pinnipedii, and M. canettii.M. tuberculosis causes the majority of clinically observed TB cases in humans and is divided into five sub-lineages termed L1-L4 and L7. M. africanum causes TB in humans and comprises lineages L5 and L6 . Wh. Wh90]. M. tuberculosis uses Mce transporters to import lipid substrates across the bacterial cell wall . Th. ThM. tue below) ,112,113.M. tuberculosis is not known to produce B12 in vitro or in macrophages suggesting the bacteria assimilates B12 or a B12 precursor directly from the host [M. canettii, has every gene required to synthesize B12 while human- and animal-adapted isolates in the MTBC lack the B12 biosynthetic enzyme, CobF [The MCC assimilates propionyl-CoA into central metabolism via succinate and pyruvate, which occurs in a vitamin B12 (B12)-independent manner. In contrast, the MMP assimilates propionyl-CoA as succinyl-CoA in a B12-dependent manner . Bacterithe host . The soime, CobF . Thus, tM. tuberculosis if the bacterium is unable to properly assimilate this intermediate [M. tuberculosis produces excess PDIM and SL as a result of coupling cholesterol metabolism to the synthesis of these lipids [M. tuberculosis during infection.Cholesterol degradation produces excess propionyl-CoA, which induce a metabolic toxicity in rmediate ,113. Shurmediate . Throughrmediate . During e lipids ,118,119.M. africanum isolates from L6 represses genes associated with PDIM synthesis during infection in macrophages [M. tuberculosis do not produce this modified PDIM due to a mutation in pks15/1 [PDIM has been implicated in macrophage invasion and recruitment ,121, resrophages . The maj pks15/1 . The gly pks15/1 , and thi pks15/1  and/or t pks15/1 ,131.M. tuberculosis metabolism. As mentioned above, cholesterol metabolism is associated with a metabolic toxicity and M. tuberculosis mitigates this by synthesizing methyl-branched polyketide lipids. Importantly the bacterium’s ability to synthesize methyl-branched lipids is limited by the amount of available fatty acid primers. For example, cholesterol-mediated toxicity in an M. tuberculosis mutant lacking Icl1 is reversed by supplying excess fatty acids to infected macrophages and these fatty acids become incorporated into PDIM [M. tuberculosis likely co-metabolizes fatty acids and cholesterol such that fatty acids are available as a “sink” for excess propionyl-CoA assimilation in the form of methyl-branched lipids such as PDIM. Thus, an important aspect of M. tuberculosis metabolism is to balance fatty acid and cholesterol utilization during infection.Recent studies have revealed a “codependency” of fatty acids and cholesterol in nto PDIM . Thus, MM. tuberculosis primarily resides in bone marrow derived, interstitial macrophages and, within alveolar macrophages that arise from the fetal liver during embryonic development [M. tuberculosis also resides in macrophages that express the M1 and M2 activation markers [Macrophages develop from distinct cell lineages and evidence is accumulating suggesting that developmental origin influences how these cells ultimately respond to infection and injury ,133. In elopment . The intelopment . Additio markers .M. tuberculosis during infection vary with the host cell and the flexible metabolism of the bacterium may allow adaptation and survival within different macrophage cell types [M. tuberculosis may benefit from the increased import of lipids by the macrophage [M. tuberculosis may preferentially metabolize glycolysis end products such as lactate that accumulate in these cells [M. tuberculosis fuels energy producing pathways in M1-like macrophages but ex vivo studies with human monocyte derived macrophages have demonstrated that M. tuberculosis can sustain a gluconeogenic pathway by assimilating lactate [Importantly, these murine macrophage subsets have highly polarized metabolic states, where the M1-like macrophages are actively glycolytic and the M2-like macrophages are actively undergoing fatty acid oxidation . This sull types . It is pcrophage . Perhapsse cells ,136. It  lactate . Going fM. tuberculosis assimilates and metabolizes nitrogen during infection in macrophages. When cultured in vitro, M. tuberculosis demonstrates a preference for amino acids as a source of nitrogen over ammonia chloride, and M. tuberculosis also has the ability to co-metabolize multiple amino acids simultaneously [M. tuberculosis encounters nitrogen in two forms: As a nitrate derived from NO and as individual amino acids. In activated macrophages, M. tuberculosis can reduce nitrate (NO3−) to nitrite (NO2−) through the activity of the multi-subunit nitrate reductase, NarGHJI [M. tuberculosis can also assimilate nitrogen from amino acids and when infected macrophages are pulsed with 15N-labeled amino acids, both aspartate and asparagine accumulate in the bacterial phagosome and the bacteria require these amino acids to maintain infections [13C-glucose established that a portion of the 13C-label accumulates in host cell amino acids and when these cells are infected M. tuberculosis is able to scavenge the13C-labeled amino acids [All cells require nitrogen to synthesize amino acids, nucleotides, and various essential cofactors. Recent studies have begun to shed light on how aneously . During  NarGHJI ,140. By  NarGHJI . M. tubefections ,143. Addno acids .M. tuberculosis from animal-adapted members of the MTBC. M. tuberculosis possesses high levels of nitrate reductase activity relative to MTBC isolates that rarely infect humans, which corresponds to promoter mutations upstream of the nitrate reductase operon in these isolates [M. tuberculosis, nitrate reductase activity has been associated with mitigating acid and reactive nitrogen stresses under hypoxic conditions [Nitrate reductase activity has been used for over 50 years in diagnostic mycobacteriology to distinguish isolates . A recenisolates . This fiisolates . In M. tnditions , howevernditions ,148. IntM. tuberculosis requires metals such as iron and copper for growth, and these elements are linked to growth and pathogenicity of M. tuberculosis in macrophages [M. tuberculosis induces the expression of many genes associated with acquiring and sequestering iron [M. tuberculosis acquires iron via siderophore scavenging from iron-loaded transferrin in the endocytic pathway [M. tuberculosis containing phagosomes have high concentrations of iron, a property that was dependent on the production of siderophores and did not occur in nonpathogenic mycobacteria [M. tuberculosis metabolism because it forms the nuclear center of the aa3-type cytochrome c oxidase [M. tuberculosis that are known to require copper for activity and this cytochrome oxidase is critical for oxidative respiration. Copper also accumulates in the phagosomes of infected macrophages, which presumably provides the bacterium necessary levels of this metal [M. tuberculosis, it appears that excess copper is associated with adverse effects on M. tuberculosis, and the bacterium has several ways to remove or inactivate this metal to avoid toxicity (see below).rophages . Iron ising iron ,75,150.  pathway ,53 or by pathway . Metal cbacteria . Similar oxidase ,153. Thiis metal . While iM. tuberculosis genome encodes numerous putative MoCo requiring enzymes, including the nitrate reductase NarGHJI [M. tuberculosis metabolism during infection remains unknown, the expansion of genes involved in MoCo is a unique feature of MTBC pathogens [M. tuberculosis is a complicated and poorly-understood process that likely involves multiple redundant enzymes [moaA1-D1) was likely acquired by M. tuberculosis during an ancient horizontal gene transfer event [M. tuberculosis during hypoxia [M. orygis isolates in the clade A3 carry a unique 13 gene deletion mutation (RD12oryx) that disables multiple putative MoCo biosynthetic genes and a transcriptional regulator of the moaA1-D1 operon [MoCo is a cyclic redox cofactor required by a subset of enzymes to catalyze carbon, nitrogen, and sulfur metabolic reactions across all kingdoms of life . The M.  NarGHJI  along wi NarGHJI . Althougathogens . The bio enzymes . A subseer event ,159,160  hypoxia . Interes1 operon ,161. It M. tuberculosis and immunologic pressures imposed by macrophages modulate the bacterium’s ability to engage metabolic pathways and utilize host-derived nutrients. Immune activation induces expression of the macrophage immunoresponsive gene-1 (Irg1 or aconitate decarboxylase 1) [M. tuberculosis and macrophage metabolism. Itaconic acid inhibits Icl1 in M. tuberculosis, which limits the bacterium’s ability to utilize intermediates from cholesterol and fatty acid degradation [M. tuberculosis compensates for this by synthesizing its own tryptophan [M. bovis increases overexpression of Ido1 although this bacterium also synthesizes its own tryptophan [Macrophages are not a static niche that simply supplies nutrients for ylase 1) . This enylase 1)  leading radation . Itaconiradation . Amino aradation . The hosyptophan ,166. Simyptophan . Given tM. tuberculosis physiology these metals also constitute an important cellular defense when these ions accumulate within macrophage lysosomes during infection [M. tuberculosis either actively secretes copper [M. tuberculosis mitigates zinc poisoning by actively exporting the metal [M. tuberculosis is equipped to mitigate copper and zinc toxicity during infection in macrophages is a relative recent finding. It will be exciting to determine how other MTBC members adapt to these metals during infection.Macrophages also possess an armamentarium of metal-based mechanisms to eliminate intracellular pathogens . Althougnfection . The P-tnfection . To mitis copper ,171, or s copper . Macrophs copper  and M. the metal . The undM. tuberculosis senses and adapts to the environmental conditions within macrophage phagosomes [M. tuberculosis is fluctuating acidity [M. tuberculosis senses and adapts to low pH environments via the two-component regulator system, PhoPR [agosomes  and one  acidity . M. tubem, PhoPR . The Phom, PhoPR , virulenm, PhoPR ,178, andm, PhoPR ,179.M. africanum and MTBC isolates that rarely infect humans have a mutation in the sensor domain of the PhoR kinase (PhoR G71I), which disables this protein [M. africanum lineage L6 and related isolates 181. Isolates in M. africanum lineage L5 also carry a non-functional PhoR but these bacteria do not have a RD8 mutation. M. africanum lineage L5 bacteria are still able to express and secrete virulence proteins (Esx) indicating that an unidentified mechanism compensates for the PhoR mutation in this lineage [Most human-adapted isolates of the MTBC have a functional PhoPR system while  protein . It is w protein . Specifi lineage .M. tuberculosis [M. tuberculosis to promote pathogenesis in macrophages.Methyl-branched lipid biosynthesis is also regulated by the PhoPR system and these lipids have long been associated with virulence in rculosis . Additiorculosis . It is tM. bovis isolates rarely transmit between humans, with a notable exception being the M. bovis isolate strain B. This strain of M. bovis efficiently transmits between humans and has been associated with high mortality among HIV-infected persons [6110 insertion upstream of phoP that enhances transcription of phoP [M. bovis this mutation allowed the bacteria to produce SL, a methyl-branched polyketide lipid [These methyl-branched polyketide lipids may also play a role in transmission. Specifically,  persons . Molecul of phoP . Importade lipid . This obM. tuberculosis to survive in macrophages because this is a common survival strategy used by MTBC pathogens that rarely infects humans. Comparative genomic approaches across members of the MTBC revealed several key differences regarding virulence and immunogenicity. Similarly, we are optimistic that comparative approaches focused on metabolism will enhance our understanding of this important group of pathogens and the animals they infect.Differences clearly exist between the MTBC members with respect to pathogenesis, host range, and transmission there are also similarities with these pathogens. This review focused on the specialized metabolism that allows