A novel symbiovar (aegeanense) of the genus Ensifer nodulates Vigna unguiculata.

BACKGROUND: Cowpea (Vigna unguiculata) forms nitrogen-fixing root nodules with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium, although a few studies have reported the isolation of fast-growing rhizobia under laboratory and field conditions. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, very limited information is available on cowpea rhizobia in European soils. The aim of this study was to study the genetic and phenotypic diversity of indigenous cowpea-nodulating rhizobia in Greece. RESULTS: The genetic diversity of indigenous rhizobia associated with cowpea was investigated through a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into three groups. Based on the analysis of the 16S rRNA genes, IGS and on the concatenation of six housekeeping genes (recA, glnII, gyrB, truA, thrA and SMc00019), rhizobial isolates were classified within the species Ensifer fredii. However, symbiotic gene phylogenies, based on nodC, nifH and rhcRST genes, showed that the Ensifer isolates are markedly diverged from type and reference strains of E. fredii and formed one clearly separate cluster. The E. fredii strains were able to nodulate and fix nitrogen in cowpea but not in soybean and common bean. CONCLUSION: The present study showed that cowpea is nodulated under field conditions by fast-growing rhizobia belonging to the species E. fredii. Based on the phylogenies, similarity levels of symbiotic genes and the host range, the Ensifer isolates may constitute a new symbiovar for which the name 'aegeanense' is proposed. © 2017 Society of Chemical Industry.

Genetic characterization at the species and symbiovar level of indigenous rhizobial isolates nodulating Phaseolus vulgaris in Greece.

Phaseolus vulgaris (L.), commonly known as bean or common bean, is considered a promiscuous legume host since it forms nodules with diverse rhizobial species and symbiovars. Most of the common bean nodulating rhizobia are mainly affiliated to the genus Rhizobium, though strains belonging to Ensifer, Pararhizobium, Mesorhizobium, Bradyrhizobium, and Burkholderia have also been reported. This is the first report on the characterization of bean-nodulating rhizobia at the species and symbiovar level in Greece. The goals of this research were to isolate and characterize rhizobia nodulating local common bean genotypes grown in five different edaphoclimatic regions of Greece with no rhizobial inoculation history. The genetic diversity of the rhizobial isolates was assessed by BOX-PCR and the phylogenetic affiliation was assessed by multilocus sequence analysis (MLSA) of housekeeping and symbiosis-related genes. A total of fifty fast-growing rhizobial strains were isolated and representative isolates with distinct BOX-PCR fingerpriniting patterns were subjected to phylogenetic analysis. The strains were closely related to R. anhuiense, R. azibense, R. hidalgonense, R. sophoriradicis, and to a putative new genospecies which is provisionally named as Rhizobium sp. I. Most strains belonged to symbiovar phaseoli carrying the α-, γ-a and γ-b alleles of nodC gene, while some of them belonged to symbiovar gallicum. To the best of our knowledge, it is the first time that strains assigned to R. sophoriradicis and harbored the γ-b allele were found in European soils. All strains were able to re-nodulate their original host, indicating that they are true microsymbionts of common bean.

Genetic diversity and phylogeny of indigenous rhizobia nodulating faba bean (Vicia faba L.) in Greece.

The genetic diversity and phylogeny of fast-growing rhizobia isolated from root nodules of Vicia faba grown in different geographical regions of Greece were assessed. Although Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia spp. in European soils, there is no available information on native rhizobia nodulating faba bean in Greece. Seventy bacterial strains were isolated and grouped into sixteen distinct profiles based on BOX-PCR fingerprinting. The phylogenetic affiliation was further defined by sequence analysis of the rrs and multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and gyrB). Fifty-eight isolates were affiliated with recently described genospecies gsF-2, represented by R. laguerreae FB206T, whereas six isolates were closely related to gsB and two isolates might belong to gsA. Two isolates assigned to R. hidalgonense and another two non-nodulating strains could not be assigned to any validly defined species and possibly belong to a new rhizobial lineage. Interestingly, R. laguerreae strains were commonly found at all sampling sites, suggesting that they could be the main symbionts of faba beans in Greek soils. According to the phylogenies of two symbiosis-related genes (nodC and nifH), all nodulating isolates belonged to symbiovar (sv.) viciae harboring four distinct nodC gene haplotypes and they were grouped into two clades together with strains assigned to R. laguerreae and genospecies of R. leguminosarum isolated from other countries and continents. This is the first report that R. hidalgonense strains belong to sv. viciae. No correlation was observed between the nodC haplotypes, geographic origin and chromosomal background of the isolates in the study.

Phylogenetic multilocus sequence analysis of indigenous slow-growing rhizobia nodulating cowpea (Vigna unguiculata L.) in Greece.

Cowpea (Vigna unguiculata) is a promiscuous grain legume, capable of establishing efficient symbiosis with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, little is known about the genetic and symbiotic diversity of indigenous cowpea rhizobia in European soils. In the present study, the genetic and symbiotic diversity of indigenous rhizobia isolated from field-grown cowpea nodules in three geographically different Greek regions were studied. Forty-five authenticated strains were subjected to a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into seven groups and representative strains of each group were further analyzed. The analysis of the rrs gene showed that the strains belong to different species of the genus Bradyrhizobium. The analysis of the 16S-23S IGS region showed that the strains from each geographic region were characterized by distinct IGS types which may represent novel phylogenetic lineages, closely related to the type species of Bradyrhizobium pachyrhizi, Bradyrhizobium ferriligni and Bradyrhizobium liaoningense. MLSA analysis of three housekeeping genes (recA, glnII, and gyrB) showed the close relatedness of our strains with B. pachyrhizi PAC48T and B. liaoningense USDA 3622T and confirmed that the B. liaoningense-related isolate VUEP21 may constitute a novel species within Bradyrhizobium. Moreover, symbiotic gene phylogenies, based on nodC and nifH genes, showed that the B. pachyrhizi-related isolates belonged to symbiovar vignae, whereas the B. liaoningense-related isolates may represent a novel symbiovar.

Image analysis driven single-cell analytics for systems microbiology.

BACKGROUND: Time-lapse microscopy is an essential tool for capturing and correlating bacterial morphology and gene expression dynamics at single-cell resolution. However state-of-the-art computational methods are limited in terms of the complexity of cell movies that they can analyze and lack of automation. The proposed Bacterial image analysis driven Single Cell Analytics (BaSCA) computational pipeline addresses these limitations thus enabling high throughput systems microbiology. RESULTS: BaSCA can segment and track multiple bacterial colonies and single-cells, as they grow and divide over time (cell segmentation and lineage tree construction) to give rise to dense communities with thousands of interacting cells in the field of view. It combines advanced image processing and machine learning methods to deliver very accurate bacterial cell segmentation and tracking (F-measure over 95%) even when processing images of imperfect quality with several overcrowded colonies in the field of view. In addition, BaSCA extracts on the fly a plethora of single-cell properties, which get organized into a database summarizing the analysis of the cell movie. We present alternative ways to analyze and visually explore the spatiotemporal evolution of single-cell properties in order to understand trends and epigenetic effects across cell generations. The robustness of BaSCA is demonstrated across different imaging modalities and microscopy types. CONCLUSIONS: BaSCA can be used to analyze accurately and efficiently cell movies both at a high resolution (single-cell level) and at a large scale (communities with many dense colonies) as needed to shed light on e.g. how bacterial community effects and epigenetic information transfer play a role on important phenomena for human health, such as biofilm formation, persisters' emergence etc. Moreover, it enables studying the role of single-cell stochasticity without losing sight of community effects that may drive it.

Molecular determinants required for the avirulence function of AvrPphB in bean and other plants.

The avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola determines incompatibility, manifested as a hypersensitive reaction (HR), on bean cultivars carrying the R3 resistance gene and also confers avirulence on other plants. The AvrPphB protein carries an embedded consensus myristoylation motif and is cleaved in bacteria and certain plants to yield fragments of about 6 and 28 kDa. We investigated plant recognition and type III translocation determinants in AvrPphB by constructing three N-terminally truncated and two site-directed mutants carrying substitutions in the conserved G63 residue of the myristoylation motif, which lies adjacent to the proteolytic cleavage site. The peptides were either delivered to plant cells by pseudomonads or were expressed transiently in planta via the Agrobacterium tumefaciens or Potato virus X. The 63 amino terminal residues were required for type III-mediated translocation from Pseudomonas strains to the plant, but were partially dispensable for effector recognition following in planta expression. Substitution of the G63 residue resulted in differential HR phenotypes in two different R3 cultivars of bean and abolished effector processing in Pseudomonas strains. Agrobacterium-mediated expression of the mutant proteins elicited HR in resistant bean hosts and in tomato but elicited no reaction in Nicotiana species.

Commonalities and differences of T3SSs in rhizobia and plant pathogenic bacteria.

Plant pathogenic bacteria and rhizobia infect higher plants albeit the interactions with their hosts are principally distinct and lead to completely different phenotypic outcomes, either pathogenic or mutualistic, respectively. Bacterial protein delivery to plant host plays an essential role in determining the phenotypic outcome of plant-bacteria interactions. The involvement of type III secretion systems (T3SSs) in mediating animal- and plant-pathogen interactions was discovered in the mid-80's and is now recognized as a multiprotein nanomachine dedicated to trans-kingdom movement of effector proteins. The discovery of T3SS in bacteria with symbiotic lifestyles broadened its role beyond virulence. In most T3SS-positive bacterial pathogens, virulence is largely dependent on functional T3SSs, while in rhizobia the system is dispensable for nodulation and can affect positively or negatively the mutualistic associations with their hosts. This review focuses on recent comparative genome analyses in plant pathogens and rhizobia that uncovered similarities and variations among T3SSs in their genetic organization, regulatory networks and type III secreted proteins and discusses the evolutionary adaptations of T3SSs and type III secreted proteins that might account for the distinguishable phenotypes and host range characteristics of plant pathogens and symbionts.

Characterization of the biocontrol activity of pseudomonas fluorescens strain X reveals novel genes regulated by glucose.

Pseudomonas fluorescens strain X, a bacterial isolate from the rhizosphere of bean seedlings, has the ability to suppress damping-off caused by the oomycete Pythium ultimum. To determine the genes controlling the biocontrol activity of strain X, transposon mutagenesis, sequencing and complementation was performed. Results indicate that, biocontrol ability of this isolate is attributed to gcd gene encoding glucose dehydrogenase, genes encoding its co-enzyme pyrroloquinoline quinone (PQQ), and two genes (sup5 and sup6) which seem to be organized in a putative operon. This operon (named supX) consists of five genes, one of which encodes a non-ribosomal peptide synthase. A unique binding site for a GntR-type transcriptional factor is localized upstream of the supX putative operon. Synteny comparison of the genes in supX revealed that they are common in the genus Pseudomonas, but with a low degree of similarity. supX shows high similarity only to the mangotoxin operon of Ps. syringae pv. syringae UMAF0158. Quantitative real-time PCR analysis indicated that transcription of supX is strongly reduced in the gcd and PQQ-minus mutants of Ps. fluorescens strain X. On the contrary, transcription of supX in the wild type is enhanced by glucose and transcription levels that appear to be higher during the stationary phase. Gcd, which uses PQQ as a cofactor, catalyses the oxidation of glucose to gluconic acid, which controls the activity of the GntR family of transcriptional factors. The genes in the supX putative operon have not been implicated before in the biocontrol of plant pathogens by pseudomonads. They are involved in the biosynthesis of an antimicrobial compound by Ps. fluorescens strain X and their transcription is controlled by glucose, possibly through the activity of a GntR-type transcriptional factor binding upstream of this putative operon.

High level resistance against rhizomania disease by simultaneously integrating two distinct defense mechanisms.

With the aim of achieving durable resistance against rhizomania disease of sugar beet, the employment of different sources of resistance to Beet necrotic yellow vein virus was pursued. To this purpose, Nicotiana benthamiana transgenic plants that simultaneously produce dsRNA originating from a conserved region of the BNYVV replicase gene and the HrpZ(Psph) protein in a secreted form (SP/HrpZ(Psph)) were produced. The integration and expression of both transgenes as well as proper production of the harpin protein were verified in all primary transformants and selfed progeny (T1, T2). Transgenic resistance was assessed by BNYVV-challenge inoculation on T2 progeny by scoring disease symptoms and DAS-ELISA at 20 and 30 dpi. Transgenic lines possessing single transformation events for both transgenes as well as wild type plants were included in inoculation experiments. Transgenic plants were highly resistant to virus infection, whereas in some cases immunity was achieved. In all cases, the resistant phenotype of transgenic plants carrying both transgenes was superior in comparison with the ones carrying a single transgene. Collectively, our findings demonstrate, for a first time, that the combination of two entirely different resistance mechanisms provide high level resistance or even immunity against the virus. Such a novel approach is anticipated to prevent a rapid virus adaptation that could potentially lead to the emergence of isolates with resistance breaking properties.

Functional characterization of NopT1 and NopT2, two type III effectors of Bradyrhizobium japonicum.

NopT1 and NopT2, putative type III effectors from the plant symbiotic bacterium Bradyrhizobium japonicum, are predicted to belong to a family of YopT/AvrPphB effectors, which are cysteine proteases. In the present study, we showed that both NopT1 and NopT2 indeed possess cysteine protease activity. When overexpressed in Escherichia coli, both NopT1 and NopT2 undergo autoproteolytic processing which is largely abolished in the presence of E-64, a papain family-specific inhibitor. Mutations of NopT1 disrupting either the catalytic triad or the putative autoproteolytic site reduce or markedly abolish the protease activity. Autocleavage likely occurs between residues K48 and M49, though another potential cleavage site is also possible. NopT1 also elicitis HR-like cell death when transiently expressed in tobacco plants and its cysteine protease activity is essential for this ability. In contrast, no macroscopic symptoms were observed for NopT2. Furthermore, mutational analysis provided evidence that NopT1 may undergo acylation inside plant cells and that this would be required for its capacity to elicit HR-like cell death in tobacco.

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet.

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.

Playing the "Harp": evolution of our understanding of hrp/hrc genes.

With the advent of recombinant DNA techniques, the field of molecular plant pathology witnessed dramatic shifts in the 1970s and 1980s. The new and conventional methodologies of bacterial molecular genetics put bacteria center stage. The discovery in the mid-1980s of the hrp/hrc gene cluster and the subsequent demonstration that it encodes a type III secretion system (T3SS) common to Gram negative bacterial phytopathogens, animal pathogens, and plant symbionts was a landmark in molecular plant pathology. Today, T3SS has earned a central role in our understanding of many fundamental aspects of bacterium-plant interactions and has contributed the important concept of interkingdom transfer of effector proteins determining race-cultivar specificity in plant-bacterium pathosystems. Recent developments in genomics, proteomics, and structural biology enable detailed and comprehensive insights into the functional architecture, evolutionary origin, and distribution of T3SS among bacterial pathogens and support current research efforts to discover novel antivirulence drugs.

MvfR, a key Pseudomonas aeruginosa pathogenicity LTTR-class regulatory protein, has dual ligands.

MvfR (PqsR), a Pseudomonas aeruginosa LysR-type transcriptional regulator, plays a critical role in the virulence of this pathogen. MvfR modulates the expression of multiple quorum sensing (QS)-regulated virulence factors; and the expression of the phnAB and pqsA-E genes that encode functions mediating 4-hydroxy-2-alkylquinolines (HAQs) signalling compounds biosynthesis, including 3,4-dihydroxy-2heptylquinoline (PQS) and its precursor 4-hydroxy-2-heptylquinoline (HHQ). PQS enhances the in vitro DNA-binding affinity of MvfR to the pqsA-E promoter, to suggest it might function as the in vivo MvfR ligand. Here we identify a novel MvfR ligand, as we show that HHQ binds to the MvfR ligand-binding-domain and potentiates MvfR binding to the pqsA-E promoter leading to transcriptional activation of pqsA-E genes. We show that HHQ is highly produced in vivo, where it is not fully converted into PQS, and demonstrate that it is required for MvfR-dependent gene expression and pathogenicity; PQS is fully dispensable, as pqsH-mutant cells, which produce HHI but completely lack PQS, display normal MvfR-dependent gene expression and virulence. Conversely, PQS is required for full production of pyocyanin. These results uncover a novel biological role for HHQ; and provide novel insights on MvfR activation that may aid in the development of therapies that prevent or treat P. aeruginosa infections in humans.

The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation: multiple quorum sensing-regulated genes are modulated without affecting lasRI, rhlRI or the production of N-acyl-L-homoserine lactones.

The transcriptional regulator MvfR is required for full Pseudomonas aeruginosa virulence, the function of multiple quorum sensing (QS)-regulated virulence factors and the synthesis of 4-hydroxy-2-alkylquinolines (HAQs), including the Pseudomonas quinolone signal (PQS). Here we investigate the role of MvfR in the QS circuitry and P. aeruginosa pathogenesis. We demonstrate using a combination of biochemical and molecular approaches, including transcription profiling, that MvfR is involved in the regulation of multiple P. aeruginosa QS-controlled genes without altering the expression of lasRI/rhlRI or the production of N-acyl-L-homoserine lactone (AHL) signals. Dissection of how mvfR is interwoven into the P. aeruginosa QS circuitry reveals that the MvfR system, through the essential contribution of PqsE, positively regulates a subset of genes dependant on both LasR and RhlR. Animal studies show that MvfR contributes to P. aeruginosa virulence by controlling the transcription of genes not under RhlR regulation, and that reduced virulence of a mvfR mutant is caused by the loss of pqsE expression and not only a deficiency in HAQs/PQS production. This study provides novel insights into the unique role of the MvfR system in AHL-mediated QS and further supports its importance in P. aeruginosa pathogenesis.

Structure of HrcQB-C, a conserved component of the bacterial type III secretion systems.

Type III secretion systems enable plant and animal bacterial pathogens to deliver virulence proteins into the cytosol of eukaryotic host cells, causing a broad spectrum of diseases including bacteremia, septicemia, typhoid fever, and bubonic plague in mammals, and localized lesions, systemic wilting, and blights in plants. In addition, type III secretion systems are also required for biogenesis of the bacterial flagellum. The HrcQ(B) protein, a component of the secretion apparatus of Pseudomonas syringae with homologues in all type III systems, has a variable N-terminal and a conserved C-terminal domain (HrcQ(B)-C). Here, we report the crystal structure of HrcQ(B)-C and show that this domain retains the ability of the full-length protein to interact with other type III components. A 3D analysis of sequence conservation patterns reveals two clusters of residues potentially involved in protein-protein interactions. Based on the analogies between HrcQ(B) and its flagellum homologues, we propose that HrcQ(B)-C participates in the formation of a C-ring-like assembly.

Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization.

Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F1/V1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa (monomer); II, approximately 300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, approximately 3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (>700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.