Investigation of the genetic structure of some common bean (Phaseolus vulgaris L.) commercial varieties and genotypes used as a genitor with SSR and SNP markers

Common bean is a species belonging to the Phaseolus genus of the Leguminosae family. It has economic importance due to being rich in protein, vitamin A and C, and minerals. Being one of the most cultivated species of legumes, the determination of genetic diversity in bean genotypes or populations has an important role in terms of our genetic resources. The objective of this study was to evaluate the genetic structure of 94 genotypes which were cultivated in different parts of the world and our country with SSR and SNP markers. 10 SSR loci and 73 SNP primers were used for the determination of genetic structure in commercial cultivars and breeding lines. All of the SSR and SNP loci used in the study were found to be polymorphic. A total of 89 alleles were identified for 10 SSR loci. Mean number of alleles per locus (Na = 8.9), effective allele number (Ne = 3.731), Shannon information index (I = 1.468), observed heterozygosity (Ho = 0.023), and expected heterozygosity (He = 0.654) were calculated based on SSR analysis. According to the results of Bayesian-based STRUCTURE analysis using SSR and SNP data, 94 bean genotypes were genetically divided into three main clusters. According to genetic distance based UPGMA dendrogram obtained from SNP analysis, 94 bean genotypes were divided into 2 main clusters corresponding Mesoamerican and Andean gene pools. The obtained results provide important information about the genetic structures of the studied bean cultivars and breeding lines. With the obtained results, it will be possible to develop breeding programs to develop new cultivars by using our gene resources.Tekirdag Namik Kemal University, Scientific Research Projects Unit [NKUBAP.03.YL.18.171]This study was funded by Tekirdag Namik Kemal University, Scientific Research Projects Unit (Project No: NKUBAP.03.YL.18.171)

RNA atlas of human bacterial pathogens uncovers stress dynamics linked to infection

Bacterial processes necessary for adaption to stressful host environments are potential targets for new antimicrobials. Here, we report large-scale transcriptomic analyses of 32 human bacterial pathogens grown under 11 stress conditions mimicking human host environments. The potential relevance of the in vitro stress conditions and responses is supported by comparisons with available in vivo transcriptomes of clinically important pathogens. Calculation of a probability score enables comparative cross-microbial analyses of the stress responses, revealing common and unique regulatory responses to different stresses, as well as overlapping processes participating in different stress responses. We identify conserved and species-specific 'universal stress responders', that is, genes showing altered expression in multiple stress conditions. Non-coding RNAs are involved in a substantial proportion of the responses. The data are collected in a freely available, interactive online resource (PATHOgenex). Bacterial stress responses are potential targets for new antimicrobials. Here, Avican et al. present global transcriptomes for 32 bacterial pathogens grown under 11 stress conditions, and identify common and unique regulatory responses, as well as processes participating in different stress responses.Peer reviewe

Maintaining Integrity Under Stress:Envelope Stress Response Regulation of Pathogenesis in Gram-Negative Bacteria

The Gram-negative bacterial envelope is an essential interface between the intracellular and harsh extracellular environment. Envelope stress responses (ESRs) are crucial to the maintenance of this barrier and function to detect and respond to perturbations in the envelope, caused by environmental stresses. Pathogenic bacteria are exposed to an array of challenging and stressful conditions during their lifecycle and, in particular, during infection of a host. As such, maintenance of envelope homeostasis is essential to their ability to successfully cause infection. This review will discuss our current understanding of the σE- and Cpx-regulated ESRs, with a specific focus on their role in the virulence of a number of model pathogens

Yersinia adhesins: an arsenal for infection

The Yersiniae are a group of Gram-negative coccobacilli inhabiting a wide range of habitats. The genus harbours three recognised human pathogens: Y. enterocolitica and Y. pseudotuberculosis, which both cause gastrointestinal disease, and Y. pestis, the causative agent of plague. These three organisms have served as models for a number of aspects of infection biology, including adhesion, immune evasion, evolution of pathogenic traits, and retracing the course of ancient pandemics. The virulence of the pathogenic Yersiniae is heavily dependent on a number of adhesin molecules. Some of these, such as the Yersinia adhesin A and invasin of the enteropathogenic species, and the pH 6 antigen of Y. pestis, have been extensively studied. However, genomic sequencing has uncovered a host of other adhesins present in these organisms, the functions of which are only starting to be investigated. Here, we review the current state of knowledge on the adhesin molecules present in the Yersiniae, their functions and putative roles in the infection process

Twin-arginine translocation in Yersinia : the substrates and their role in virulence

Pathogenic Yersinia cause a manifold of diseases in humans ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to pneumonic and bubonic plague (Y. pestis), while all three have a common virulence strategy that relies on a well-studied type III secretion system and its effector proteins to colonize the host and evade immune responses. However, the role of other protein secretion and/or translocation systems in virulence of Yersinia species is not well known. In this thesis, we sought to investigate the contribution of twin-arginine translocation (Tat) pathway and its secreted substrates to the physiology and virulence of Y. pseudotuberculosis. Tat pathway uniquely exports folded proteins including virulence factors across the cytoplasmic membranes of bacteria. The proteins exported by Tat pathway contain a highly conserved twin-arginine motif in the N-terminal signal peptide. We found that the loss of Tat pathway causes a drastic change of the transcriptome of Y. pseudotuberculosis in stationary phase at environmental temperature with differential regulation of genes involved in virulence, carbon metabolism and stress responses. Phenotypic analysis revealed novel phenotypes of the Tat-deficient strain with defects in iron acquisition, acid resistance, copper oxidation and envelope integrity, which we were partly able to associate with the related Tat substrates. Moreover, increased glucose consumption and accumulation of intracellular fumarate were observed in response to inactivation of Tat pathway implicating a generic effect in cellular physiology. We evaluated the direct role of 22 in silico predicted Tat substrate mutants in the mouse infection model and found only one strain, ΔsufI, exhibited a similar degree of attenuation as Tat-deficient strain. Comparative in vivo characterization studies demonstrated a minor defect for ΔsufI in colonization of intestinal tissues compared to the Tat-deficient strain during early infection, whereas both SufI and TatC were required for dissemination from mesenteric lymph nodes and further systemic spread during late infection. This verifies that SufI has a major role in attenuation seen for the Tat deficient strain both during late infection and initial colonization. It is possible that other Tat substrates such as those involved in iron acquisition and copper resistance also has a role in establishing infection. Further phenotypic analysis indicated that SufI function is required for cell division and stress-survival. Transcriptomic analysis revealed that the highest number of differentially regulated genes in response to loss of Tat and SufI were involved in metabolism and transport. Taken together, this thesis presents a thorough analysis of the involvement of Tat pathway in the overall physiology and virulence strategies of Y. pseudotuberculosis. Finally, we propose that strong effects in virulence render TatC and SufI as potential targets for development of novel antimicrobial compound

Persistent infection by Yersinia pseudotuberculosis

Enteropathogenic Yersinia species can infect many mammalian organs such as the small intestine, cecum, Peyer’s patches, liver, spleen, and lung and cause diseases that resemble a typhoid-like syndrome, as seen for other enteropathogens. We found that sublethal infection doses of Y. pseudotuberculosis gave rise to asymptomatic persistent infection in mice and identified the cecal lymphoid follicles as the primary site for colonization during persistence. Persistent Y. pseudotuberculosis is localized in the dome area, often in inflammatory lesions, as foci or as single cells, and also in neutrophil exudates in the cecal lumen. This new mouse model for bacterial persistence in cecum has potential as an investigative tool for deeper understanding of bacterial adaptation and host immune defense mechanisms during persistent infection. Here, we investigated the nature of the persistent infection established by Y. pseudotuberculosis in mouse cecal tissue using in vivo RNA-seq of bacteria during early and persistent stages of infection. Comparative analysis of the bacterial transcriptomes revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence in the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26°C. Genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, we show that ArcA, Fnr, FrdA, WrbA, RovA, and RfaH play critical roles in persistence. An extended investigation of the transcriptional regulator rfaH employing mouse infection studies, phenotypic characterizations, and RNA-seq transcriptomics analyses indicated that this gene product contributes to establishment of infection and confirmed that it regulates O-antigen biosynthesis genes in Y. pseudotuberculosis. The RNA-seq results also suggest that rfaH has a relatively global effect. Furthermore, we also found that the dynamics of the cecal tissue organization and microbial composition shows changes during different stages of the infection. Taken together, based on our findings, we speculate that this enteropathogen initiates infection by using its virulence factors in meeting the innate immune response in the cecal tissue. Later on, these factors lead to dysbiosis in the local microbiota and altered tissue organization. At later stages of the infection, the pathogen adapts to the environment in the cecum by reprogramming its transcriptome from a highly virulent mode to a more environmentally adaptable mode for survival and shedding. The in vivo transcriptomic analyses for essential genes during infections present strong candidates for novel targets for antimicrobials

Genetic characterization of some common bean (phaseolus l. ) cemmercial varieties and gentypes used as a genitor in bean breeding programs with aas and snp markers

Fasulye Leguminosae familyasından Phaseolus cinsine ait bir türdür. Fasulye içerdiği protein, A ile C vitamini ve mineral yönünden zengin olması nedeniyle ekonomik öneme sahiptir. Fasulye Dünya genelinde yetiştirilen baklagiller içerisinde en fazla ekim alanına sahip olan baklagildir. Ülkemizdeki fasulye genotip veya populasyonlarının genetik çeşitliliğinin tam olarak ortaya konması gen kaynaklarımız açısından önemli bir yere sahiptir. Bu tez çalışması kapsamında Dünya’da ve Türkiye’nin farklı bölgelerinde ekimi gerçekleştirilen ticari fasulye çeşitlerine ve ıslah materyallerine ait toplamda 94 genotip kullanılmıştır. 10 SSR lokusu (BM141, BM143, BM152, BM160, BM172, GATS91, PV-at002, PV-ctt001, PV-ag001 ve PV-at007) ve 73 polimorfik SNP primeri kullanılarak çeşit ve ıslah hatlarının genetik yapısı incelenmiştir. Çalışmada kullanılan SSR lokuslarının tamamı polimorfik olarak saptanmıştır. Analiz edilen 94 örnekte 10 SSR lokusu için toplam 89 allel saptanmıştır. SSR analizi sonucunda lokus başına düşen ortalama allel sayısı (Na=8,9), etkili allel sayısı (Ne=3,731), Shannon Sabiti (I=1,468), gözlenen heterozigotluk (Ho=0,023) ve beklenen heterozigotluk (He=0,654) hesaplanmıştır. Çalışma sonucunda SSR verileri kullanılarak yapılan Bayesian temelli STRUCTURE analizi sonucuna göre 94 fasulye genotipi genetik olarak kendi arasında 3 ana gruba ayrılmıştır. Fasulye genotiplerinin SNP analizi sonucunda genetik benzerlik değerlerine göre elde edilen UPGMA dendrogramına göre 2 ana gruba ayrılmıştır. Elde edilen sonuçlar, çalışılan fasulye çeşitlerinin ve ıslah hatlarının genetik yapıları hakkında önemli bilgiler vermiştir. Bu çalışmanın ışığında elde edilen sonuçlar ile kendi gen kaynaklarımız kullanılarak yeni çeşitler geliştirmeye yönelik ıslah programlarının planlanması mümkün olacaktır.Common bean is a species belonging to the Phaseolus genus of Leguminosae family. It has economic importance due to being rich in protein, vitamin A and C, and minerals. Among the legumes grown in the world, the bean is one of the most cultivated species of legumes. The determination of genetic diversity in bean genotypes or populations has an important role in terms of our genetic resources. In this study, 94 genotypes which were cultivated in different parts of the world and our country were investigated with SSR and SNP markers. 10 SSR locus (BM141, BM143, BM152, BM160, BM172, GATS91, PV-at002, PV-ctt001, PV-ag001, and PV-at007) and 73 polymorphic SNP primer were used for the determination of genetic structure in commercial cultivars and breeding lines. All of the SSR loci used in the study were found to be polymorphic. A total of 89 alleles were identified for 10 SSR loci in 94 samples analyzed. Mean number of alleles per locus (Na=8.9), effective allele number (Ne=3.731), Shannon information index (I=1.468), observed heterozygosity (Ho=0.023) and expected heterozygosity (He=0.654) were calculated based on SSR analysis. According to the results of Bayesian based STRUCTURE analysis using SSR data, 94 bean genotypes were genetically divided into three main groups. According to genetic similarity based UPGMA dendrogram obtained from SNP analysis, 94 bean genotypes were divided into 2 main groups. The obtained results provide important information about the genetic structures of the studied bean cultivars and breeding lines. With the significant results obtained from this thesis, it will be possible to develop breeding programs to develop new cultivars by using our own gene resources

Persistent infection by Yersinia pseudotuberculosis

Enteropathogenic Yersinia species can infect many mammalian organs such as the small intestine, cecum, Peyer’s patches, liver, spleen, and lung and cause diseases that resemble a typhoid-like syndrome, as seen for other enteropathogens. We found that sublethal infection doses of Y. pseudotuberculosis gave rise to asymptomatic persistent infection in mice and identified the cecal lymphoid follicles as the primary site for colonization during persistence. Persistent Y. pseudotuberculosis is localized in the dome area, often in inflammatory lesions, as foci or as single cells, and also in neutrophil exudates in the cecal lumen. This new mouse model for bacterial persistence in cecum has potential as an investigative tool for deeper understanding of bacterial adaptation and host immune defense mechanisms during persistent infection. Here, we investigated the nature of the persistent infection established by Y. pseudotuberculosis in mouse cecal tissue using in vivo RNA-seq of bacteria during early and persistent stages of infection. Comparative analysis of the bacterial transcriptomes revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence in the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26°C. Genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, we show that ArcA, Fnr, FrdA, WrbA, RovA, and RfaH play critical roles in persistence. An extended investigation of the transcriptional regulator rfaH employing mouse infection studies, phenotypic characterizations, and RNA-seq transcriptomics analyses indicated that this gene product contributes to establishment of infection and confirmed that it regulates O-antigen biosynthesis genes in Y. pseudotuberculosis. The RNA-seq results also suggest that rfaH has a relatively global effect. Furthermore, we also found that the dynamics of the cecal tissue organization and microbial composition shows changes during different stages of the infection. Taken together, based on our findings, we speculate that this enteropathogen initiates infection by using its virulence factors in meeting the innate immune response in the cecal tissue. Later on, these factors lead to dysbiosis in the local microbiota and altered tissue organization. At later stages of the infection, the pathogen adapts to the environment in the cecum by reprogramming its transcriptome from a highly virulent mode to a more environmentally adaptable mode for survival and shedding. The in vivo transcriptomic analyses for essential genes during infections present strong candidates for novel targets for antimicrobials

Co-PATHOgenex web application for assessing complex stress responses in pathogenic bacteria

Pathogenic bacteria encounter various stressors while residing in the host. They respond through intricate mechanisms of gene expression regulation, ensuring their survival and adaptation. Understanding how bacteria adapt to different stress conditions through regulatory processes of specific genes requires exploring complex transcriptional responses using gene co-expression networks. We employed a large transcriptome data set comprising 32 diverse human bacterial pathogens exposed to the same 11 host-mimicking stress conditions. Using the weighted gene co-expression network analysis algorithm, we generated bacterial gene co-expression networks. By associating modular eigengene expression with specific stress conditions, we identified gene co-expression modules and stress-specific stimulons, including genes with unique expression patterns under specific stress conditions. Suggesting a new potential role of the frm operon in responding to bile stress in enteropathogenic bacteria demonstrates the effectiveness of our approach. We also revealed the regulation of streptolysin S genes, involved in the production, processing, and export of streptolysin S, a toxin responsible for the beta-hemolytic phenotype of group A Streptococcus. In a comparative analysis of stress responses in three Escherichia coli strains from the core transcriptome, we revealed shared and unique expression patterns across the strains, offering insights into convergent and divergent stress responses. To help researchers perform similar analyses, we created the user-friendly web application Co-PATHOgenex. This tool aids in deepening our understanding of bacterial adaptation to stress conditions and in deciphering complex transcriptional responses of bacterial pathogens.IMPORTANCEUnveiling gene co-expression networks in bacterial pathogens has the potential for gaining insights into their adaptive strategies within the host environment. Here, we developed Co-PATHOgenex, an interactive and user-friendly web application that enables users to construct networks from gene co-expressions using custom-defined thresholds (https://avicanlab.shinyapps.io/copathogenex/). The incorporated search functions and visualizations within the tool simplify the usage and facilitate the interpretation of the analysis output. Co-PATHOgenex also includes stress stimulons for various bacterial species, which can help identify gene products not previously associated with a particular stress condition. Unveiling gene co-expression networks in bacterial pathogens has the potential for gaining insights into their adaptive strategies within the host environment. Here, we developed Co-PATHOgenex, an interactive and user-friendly web application that enables users to construct networks from gene co-expressions using custom-defined thresholds (https://avicanlab.shinyapps.io/copathogenex/). The incorporated search functions and visualizations within the tool simplify the usage and facilitate the interpretation of the analysis output. Co-PATHOgenex also includes stress stimulons for various bacterial species, which can help identify gene products not previously associated with a particular stress condition

Transcriptomic and Phenotypic Analysis Reveals New Functions for the Tat Pathway in Yersinia pseudotuberculosis

Avican U, Beckstette M, Heroven AK, Lavander M, Dersch P, Forsberg Å. Transcriptomic and Phenotypic Analysis Reveals New Functions for the Tat Pathway in Yersinia pseudotuberculosis. Journal of Bacteriology. 2016;198(20):2876-2886.The twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and ΔtatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ΔtatC mutant were seen at 26°C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. IMPORTANCE In addition to its established role in mediating the secretion of housekeeping enzymes, the Tat system has been recognized as being involved in infection. In some clinically relevant bacteria, such as Pseudomonas spp., several key virulence determinants can readily be identified among the Tat substrates. In enteropathogens, such as Yersinia spp., there are no obvious virulence determinants among the Tat substrates. Tat mutants show no growth defect in vitro but are highly attenuated in in vivo. This makes Tat an attractive target for the development of novel antimicrobials. Therefore, it is important to establish the causes of the attenuation. Here, we show that the attenuation is likely due to synergistic effects of different Tat-dependent phenotypes that each contributes to lowered in vivo fitness