Compatible Consortium of Endophytic Bacillus halotolerans Strains Cal.l.30 and Cal.f.4 Promotes Plant Growth and Induces Systemic Resistance against Botrytis cinerea.

Evaluating microbial-based alternatives to conventional fungicides and biofertilizers enables us to gain a deeper understanding of the biocontrol and plant growth-promoting activities. Two genetically distinct Bacillus halotolerans strains (Cal.l.30, Cal.f.4) were evaluated for the levels of their compatibility. They were applied individually or in combination under in vitro and greenhouse conditions, using seed bio-priming and soil drenching as inoculum delivery systems, for their plant growth-promoting effect. Our data indicate that application of Cal.l.30 and Cal.f.4 as single strains and as a mixture significantly enhanced growth parameters of Arabidopsis and tomato plants. We investigated whether seed and an additional soil treatment with these strains could induce the expression of defense-related genes in leaves of young tomato seedling plants. These treatments mediated a long lasting, bacterial-mediated, systemic-induced resistance as evidenced by the high levels of expression of RP3, ACO1 and ERF1 genes in the leaves of young tomato seedlings. Furthermore, we presented data showing that seed and soil treatment with B. halotolerans strains resulted in an effective inhibition of Botrytis cinerea attack and development on tomato leaves. Our findings highlighted the potential of B. halotolerans strains as they combine both direct antifungal activity against plant pathogens and the ability to prime plant innate immunity and enhance plant growth.

Calendula officinalis-A Great Source of Plant Growth Promoting Endophytic Bacteria (PGPEB) and Biological Control Agents (BCA).

The application of beneficial bacteria may present an alternative approach to chemical plant protection and fertilization products as they enhance growth and resistance to biotic and abiotic stresses. Plant growth-promoting bacteria are found in the rhizosphere, epiphytically or endophytically (Plant Growth Promoting Endophytic Bacteria, PGPEB). In the present study, 36 out of 119 isolated endophytic bacterial strains from roots, leaves and flowers of the pharmaceutical plant Calendula officinalis were further identified and classified into Bacillus, Pseudomonas, Pantoea, Stenotrophomonas and Rhizobium genera. Selected endophytes were evaluated depending on positive reaction to different plant growth promoting (PGP) traits, motility, survival rate and inhibition of phytopathogenic fungi in vitro and ex vivo (tomato fruit). Bacteria were further assessed for their plant growth effect on Arabidopsis thaliana seedlings and on seed bio-primed tomato plantlets, in vitro. Our results indicated that many bacterial endophytes increased seed germination, promoted plant growth and changed root structure by increasing lateral root density and length and root hair formation. The most promising antagonistic PGPEB strains (Cal.r.29, Cal.l.30, Cal.f.4, Cal.l.11, Cal.f.2.1, Cal.r.19 and Cal.r.11) are indicated as effective biological control agents (BCA) against Botrytis cinerea on detached tomato fruits. Results underlie the utility of beneficial endophytic bacteria for sustainable and efficient crop production and disease control.

Integrated Genomic and Metabolomic Analysis Illuminates Key Secreted Metabolites Produced by the Novel Endophyte Bacillus halotolerans Cal.l.30 Involved in Diverse Biological Control Activities.

The endophytic strain Cal.l.30, isolated from the medicinal plant Calendula officinalis, was selected among seven Bacillus strains with plant growth promoting activity and strong biological potential against the postharvest fungal pathogen Botrytis cinerea. Treatment by inoculating Cal.l.30 bacterial cell culture or cell free supernatant on harvested grapes and cherry tomato fruits, significantly reduced gray mold disease severity index and disease incidence. Based on 16S rRNA sequence analysis and whole genome phylogeny, Cal.l.30 was identified as Bacillus halotolerans. Genome mining revealed that B. halotolerans Cal.l.30 is endowed with a diverse arsenal of secondary metabolite biosynthetic gene clusters (SM-BGCs) responsible for metabolite production with antimicrobial properties. A sub-set of the identified SM-BGCs (mojavensin A, 'bacillunoic acid') appears to be the result of recent horizontal gene transfer events. Its genome was also mined for CAZymes associated with antifungal activity. Further UHPLC-HRMS analysis indicated that Cal.l.30 synthesizes and secretes secondary metabolites with antimicrobial activity, including the lipopeptides, fengycin, surfactin and mojavensin A, bacillaene isoforms, L-dihydroanticapsin and bacillibactin. Other compounds with known antimicrobial activity were also detected, such as azelaic acid, 15- hydroxypentadecanoid acid and 2-hydroxyphenylacetic acid. The genomic and metabolomic features of the B. halotolerans Cal.l.30 provided new perspectives on the exploitation of novel Bacillus sp. as a biocontrol agent.

Genomic and Metabolomic Insights into Secondary Metabolites of the Novel Bacillus halotolerans Hil4, an Endophyte with Promising Antagonistic Activity against Gray Mold and Plant Growth Promoting Potential.

The endophytic bacterial strain Hil4 was isolated from leaves of the medicinal plant Hypericum hircinum. It exhibited antifungal activity against Botrytis cinerea and a plethora of plant growth promoting traits in vitro. Whole genome sequencing revealed that it belongs to Bacillus halotolerans and possesses numerous secondary metabolite biosynthetic gene clusters and genes involved in plant growth promotion, colonization, and plant defense elicitation. The Mojavensin cluster was present in the genome, making this strain novel among plant-associated B. halotolerans strains. Extracts of secreted agar-diffusible compounds from single culture secretome extracts and dual cultures with B. cinerea were bioactive and had the same antifungal pattern on TLC plates after bioautography. UHPLC-HRMS analysis of the single culture secretome extract putatively annotated the consecutively produced antimicrobial substances and ISR elicitors. The isolate also proved efficient in minimizing the severity of gray mold post-harvest disease on table grape berries, as well as cherry tomatoes. Finally, it positively influenced the growth of Arabidopsis thaliana Col-0 and Solanum lycopersicum var. Chondrokatsari Messinias after seed biopriming in vitro. Overall, these results indicate that the B. halotolerans strain Hil4 is a promising novel plant growth promoting and biocontrol agent, and can be used in future research for the development of biostimulants and/or biological control agents.

Whole-genome sequence data of the proteolytic and bacteriocin producing strain Enterococcus faecalis PK23 isolated from the traditional Halitzia cheese produced in Cyprus.

Halitzia is a traditional white-brined cheese produced by a limited number of producers in Cyprus. During a survey of the microbiome of a number of different Halitzia samples, we identified a bacterial strain that exhibited enhanced proteolytic activity compared to the other isolates. The strain was further studied, and it was assigned as Enterococcus faecalis PK23. We proceeded with sequencing of its whole genome using Illumina technology. Initial sequencing and assembly produced 116 scaffolds with a length of 3,149,036 bp. Comparison with the available E. faecalis genomes revealed that the strain PK23 exhibited high levels of identity to the genome sequence of E. faecalis isolate 26975_2#180 deposited in GenBank as a single complete contig. From the 116 scaffolds 106 could be aligned to the genome of isolate 26975_2#180 leading to a chromosomal length of 3,132,784 bp with a GC content of 37.3%. From the remaining 10 scaffolds, five showed similarity to plasmid sequences. More specifically, scaffold 54 showed high identity with most part of plasmid pEF1071 of E. faecalis strain BFE 1071, which carries the gene cluster involved in the biosynthesis of enterocins 1071A and 1071B, while scaffold 77 showed high identity with the entire sequence of the unnamed_5 cryptic plasmid of Enterococcus faecium strain PR05720-3. The other three scaffolds were only short parts of larger plasmids. The remaining five scaffolds which could not be related to any plasmid sequence most probably constitute chromosomal sequences present in strain PK23 but absent from isolate 26975_2#180. Their total length was around 2.7 kb, which does not affect the sequence of the PK23 pseudochromosome in a major way. The whole-genome sequence annotation of strain PK23 identified 3161 coding sequences and 62 RNA sequences. The results from the Rapid Annotation using Subsystem Technology (RAST) version 2.0 server indicated the presence of seven putative genes which were related to the subsystem of Protein Degradation. This dataset provides a first overview of the proteolytic and bacteriocin producing properties of E. faecalis PK23. The dataset may also be used in future experiments which could shed light on the adaptation of the strain in the dairy environment and its role in cheese production.

Genomic Analysis and Secondary Metabolites Production of the Endophytic Bacillus velezensis Bvel1: A Biocontrol Agent against Botrytis cinerea Causing Bunch Rot in Post-Harvest Table Grapes.

Botrytis bunch rot caused by Botrytis cinerea is one of the most economically significant post-harvest diseases of grapes. In the present study, we showed that the bacterial strain Bvel1 is phylogenetically affiliated to Bacillus velezensis species. The strain Bvel1 and its secreted metabolites exerted an antifungal activity, under in vitro conditions, against B. cinerea. UHPLC-HRMS chemical analysis revealed that iturin A2, surfactin-C13 and -C15, oxydifficidin, bacillibactin, L-dihydroanticapsin, and azelaic acid were among the metabolites secreted by Bvel1. Treatment of wounded grape berries with Bacillus sp. Bvel1 cell culture was effective for controlling grey mold ingress and expansion in vivo. The effectiveness of this biological control agent was a function of the cell culture concentration of the antagonist applied, while preventive treatment proved to be more effective compared to curative. The strain Bvel1 exhibited an adequate colonization efficiency in wounded grapes. The whole-genome phylogeny, combined with ANI and dDDH analyses, provided compelling evidence that the strain Bvel1 should be taxonomically classified as Bacillus velezensis. Genome mining approaches showed that the strain Bvel1 harbors 13 antimicrobial biosynthetic gene clusters, including iturin A, fengycin, surfactin, bacilysin, difficidin, bacillaene, and bacillibactin. The results provide new insights into the understanding of the endophytic Bacillus velezensis Bvel1 biocontrol mechanism against post-harvest fungal pathogens, including bunch rot disease in grape berries.

Multi-Trait Wheat Rhizobacteria from Calcareous Soil with Biocontrol Activity Promote Plant Growth and Mitigate Salinity Stress.

Plant growth promoting rhizobacteria (PGPR) can be functional microbial fertilizers and/or biological control agents, contributing to an eco-spirit and safe solution for chemical replacement. Therefore, we have isolated rhizospheric arylsulfatase (ARS)-producing bacteria, belonging to Pseudomonas and Bacillus genus, from durum wheat crop grown on calcareous soil. These isolates harbouring plant growth promoting (PGP) traits were further evaluated in vitro for additional PGP traits, including indole compounds production and biocontrol activity against phytopathogens, limiting the group of multi-trait strains to eight. The selected bacterial strains were further evaluated for PGP attributes associated with biofilm formation, compatibility, salt tolerance ability and effect on plant growth. In vitro studies demonstrated that the multi-trait isolates, Bacillus (1.SG.7, 5.SG.3) and Pseudomonas (2.SG.20, 2.C.19) strains, enhanced the lateral roots abundance and shoots biomass, mitigated salinity stress, suggesting the utility of beneficial ARS-producing bacteria as potential microbial fertilizers. Furthermore, in vitro studies demonstrated that compatible combinations of multi-trait isolates, Bacillus sp. 1.SG.7 in a mixture coupled with 5.SG.3, and 2.C.19 with 5.SG.3 belonging to Bacillus and Pseudomonas, respectively, may enhance plant growth as compared to single inoculants.

Impact of Elemental Sulfur on the Rhizospheric Bacteria of Durum Wheat Crop Cultivated on a Calcareous Soil.

Previous experiments have shown that the application of fertilizer granules containing elemental sulfur (S0) as an ingredient (FBS0) in durum wheat crops produced a higher yield than that produced by conventional ones (F), provided that the soils of the experimental fields (F vs. FBS0) were of comparable quality and with the Olsen P content of the field's soil above 8 mg kg-1. In this experiment the FBS0 treatment took place in soil with Olsen P at 7.8 mg kg-1, compared with the F treatment's soil with Olsen P of 16.8 mg kg-1, aiming at reducing the imbalance in soil quality. To assess and evaluate the effect of FBS0 on the dynamics of the rhizospheric bacteria in relation to F, rhizospheric soil at various developmental stages of the crops was collected. The agronomic profile of the rhizospheric cultivable bacteria was characterized and monitored, in connection with the dynamics of phosphorus, iron, organic sulfur, and organic nitrogen, in both the rhizosoil and the aerial part of the plant during development. Both crops were characterized by a comparable dry mass accumulation per plant throughout development, while the yield of the FBS0 crop was 3.4% less compared to the F crop's one. The FBS0 crop's aerial part showed a transient higher P and Fe concentration, while its organic N and S concentrations followed the pattern of the F crop. The incorporation of S0 into the conventional fertilizer increased the percentage of arylsulfatase (ARS)-producing bacteria in the total bacterial population, suggesting an enhanced release of sulfate from the soil's organic S pool, which the plant could readily utilize. The proportion of identified ARS-producing bacteria possessing these traits exhibited a maximum value before and after topdressing. Phylogenetic analysis of the 68 isolated ARS-producing bacterial strains revealed that the majority of the isolates belonged to the Pseudomonas genus. A large fraction also possessed phosphate solubilization, and/or siderophore production, and/or ureolytic traits, thus improving the crop's P, Fe, S, and N balance. The aforementioned findings imply that the used FBS0 substantially improved the quality of the rhizosoil at the available phosphorus limiting level by modulating the abundance of the bacterial communities in the rhizosphere and effectively enhancing the microbially mediated nutrient mobilization towards improved plant nutritional dynamics.

Structure-Function Analysis of the Periplasmic Escherichia coli Cyclophilin PpiA in Relation to Biofilm Formation.

The presence of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8) in all domains of life indicates their biological importance. Cyclophilin PpiA, present in the periplasm of gram-negative bacteria, possesses PPIase activity but its physiological functions are still not clearly defined. Here, we demonstrate that the ΔppiA deletion strain from Escherichia coli exhibits an increased ability for biofilm formation and enhanced swimming motility compared to the wild-type strain. To identify structural features of PpiA which are necessary for the negative modulation of biofilm formation, we constructed a series of mutant PpiA proteins using a combination of error-prone and site-directed mutagenesis approaches. We show that the negative effect of PpiA on biofilm formation is not dependent on its PPIase activity, since PpiA mutants with a reduced PPIase activity are able to complement the ΔppiA strain during biofilm growth.

Microbial cyclophilins: specialized functions in virulence and beyond.

Cyclophilins belong to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), the enzymes that catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds in unfolded and partially folded polypeptide chains and native state proteins. Cyclophilins have been extensively studied, since they are involved in multiple cellular processes related to human pathologies, such as neurodegenerative disorders, infectious diseases, and cancer. However, the presence of cyclophilins in all domains of life indicates a broader biological importance. In this mini-review, we summarize current advances in the study of microbial cyclophilins. Apart from their anticipated role in protein folding and chaperoning, cyclophilins are involved in several other biological processes, such as cellular signal transduction, adaptation to stress, control of pathogens virulence, and modulation of host immune response. Since many existing family members do not have well-defined functions and novel ones are being characterized, the requirement for further studies on their biological role and molecular mechanism of action is apparent.

Functional analysis of the cyclophilin PpiB role in bacterial cell division.

Escherichia coli PpiB is a peptidyl-prolyl cis/trans isomerase (PPIase, EC: 5.2.1.8) with chaperone activity. Here, we show that the ΔppiB deletion strain and the PpiB over-expression wild-type strain are both characterized by defects in cell division involving milder or severe cell filamentation, respectively. Using various PpiB mutants, we show that the PPIase activity of PpiB is necessary for the observed cell filamentation, whereas other structural features apart from the active site are also important for this phenotype. Early divisome components zipA and ftsZ showed decreased expression in ΔppiB cells, whereas the corresponding proteins partially suppressed the division phenotype of ΔppiB cells as well. Although PpiB itself has no obvious specific affinity for the septal ring as a GFP translational fusion showed a diffuse cytoplasmic localization, it interacts with FtsZ employing the C-terminal FtsZ domain, decreases its GTPase activity and when over-expressed shows an inhibitory effect on the proper FtsZ localization at future division sites. Furthermore, additional putative PpiB prey proteins are able to partially restore the ΔppiB phenotype indicating that PpiB is able to control bacterial cell division by probably modulating the function of various other proteins which are indirectly associated with the process.

Functional analysis of the two cyclophilin isoforms of Sinorhizobium meliloti.

The nitrogen fixing Sinorhizobium meliloti possesses two genes, ppiA and ppiB, encoding two cyclophilin isoforms which belong to the superfamily of peptidyl prolyl cis/trans isomerases (PPIase, EC: 5.2.1.8). Here, we functionally characterize the two proteins and we demonstrate that both recombinant cyclophilins are able to isomerise the Suc-AAPF-pNA synthetic peptide but neither of them displays chaperone function in the citrate synthase thermal aggregation assay. Furthermore, we observe that the expression of both enzymes increases the viability of E. coli BL21 in the presence of abiotic stress conditions such as increased heat and salt concentration. Our results support and strengthen previous high-throughput studies implicating S. meliloti cyclophilins in various stress conditions.

Structural and functional analysis of cyclophilin PpiB mutants supports an in vivo function not limited to prolyl isomerization activity.

Escherichia coli cyclophilin PpiB is a peptidyl-prolyl cis/trans isomerase (PPIase, EC: 5.2.1.8), involved in the negative modulation of various bacterial processes, such as swimming and swarming motility and biofilm formation ability. In this study, we show that PpiB possesses also a chaperone function as it can prevent the thermal denaturation of citrate synthase even with essentially eliminated PPIase activity. We demonstrate, using active site mutations, that the PPIase activity of PpiB is required in all processes, except for the negative effect on swimming, indicating a possible isomerase-independent function. Additionally, we show that the reduced PPIase activity of PpiB does not prevent the association with all prey proteins tested and that the PPIase active site is not involved necessarily in each association. We also used a random mutagenesis approach, to identify amino acid residues apart from the catalytic site, which are necessary for PpiB function. The combination of enzymatic studies concerning the PPIase and chaperone activities of each mutant protein, with structural analyses based on 3D models, provided further insights into the effects of the mutations on the function of PpiB and showed the importance of structural features in addition to the catalytic site, for its in vivo role.

Cyclophilin PpiB is involved in motility and biofilm formation via its functional association with certain proteins.

PpiB belongs to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), which catalyze the rate-limiting protein folding step at peptidyl-prolyl bonds and control several biological processes. In this study, we show that PpiB acts as a negative effector of motility and biofilm formation ability of Escherichia coli. We identify multicopy suppressors of each ΔppiB phenotype among putative PpiB prey proteins which upon deletion are often characterized by analogous phenotypes. Many putative preys show similar gene expression in wild-type and ΔppiB genetic backgrounds implying possible post-translational modifications by PpiB. We further conducted in vivo and in vitro interaction screens to determine which of them represent true preys. For DnaK, acetyl-CoA carboxylase, biotin carboxylase subunit (AccC) and phosphate acetyltransferase (Pta) we also showed a direct role of PpiB in the functional control of these proteins because it increased the measured enzyme activity of each protein and further interfered with DnaK localization and the correct folding of AccC. Taken together, these results indicate that PpiB is involved in diverse regulatory mechanisms to negatively modulate motility and biofilm formation via its functional association with certain protein substrates.

The nitrogen-fixation island insertion site is conserved in diazotrophic Pseudomonas stutzeri and Pseudomonas sp. isolated from distal and close geographical regions.

The presence of nitrogen fixers within the genus Pseudomonas has been established and so far most isolated strains are phylogenetically affiliated to Pseudomonas stutzeri. A gene ortholog neighborhood analysis of the nitrogen fixation island (NFI) in four diazotrophic P. stutzeri strains and Pseudomonas azotifigens revealed that all are flanked by genes coding for cobalamin synthase (cobS) and glutathione peroxidise (gshP). The putative NFIs lack all the features characterizing a mobilizable genomic island. Nevertheless, bioinformatic analysis P. stutzeri DSM 4166 NFI demonstrated the presence of short inverted and/or direct repeats within both flanking regions. The other P. stutzeri strains carry only one set of repeats. The genetic diversity of eleven diazotrophic Pseudomonas isolates was also investigated. Multilocus sequence typing grouped nine isolates along with P. stutzeri and two isolates are grouped in a separate clade. A Rep-PCR fingerprinting analysis grouped the eleven isolates into four distinct genotypes. We also provided evidence that the putative NFI in our diazotrophic Pseudomonas isolates is flanked by cobS and gshP genes. Furthermore, we demonstrated that the putative NFI of Pseudomonas sp. Gr65 is flanked by inverted repeats identical to those found in P. stutzeri DSM 4166 and while the other P. stutzeri isolates harbor the repeats located in the intergenic region between cobS and glutaredoxin genes as in the case of P. stutzeri A1501. Taken together these data suggest that all putative NFIs of diazotrophic Pseudomonas isolates are anchored in an intergenic region between cobS and gshP genes and their flanking regions are designated by distinct repeats patterns. Moreover, the presence of almost identical NFIs in diazotrophic Pseudomonas strains isolated from distal geographical locations around the world suggested that this horizontal gene transfer event may have taken place early in the evolution.

Functional interaction of Azotobacter vinelandii cytoplasmic cyclophilin with the biotin carboxylase subunit of acetyl-CoA carboxylase.

Cyclophilins (E.C. 5.1.2.8) are protein chaperones with peptidyl-prolyl cis/trans isomerase activity (PPIase). In the present study, we demonstrate a physical interaction among AvppiB, encoding the cytoplasmic cyclophilin from the soil nitrogen-fixing bacterium Azotobacter vinelandii, and AvaccC, encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. A decrease in AvppiB PPIase activity, in the presence of AvaccC, further confirms the interaction. However, PPIase activity seems not to be essential for these interactions since a PPIase active site mutant of cyclophilin does not abolish the AvaccC binding. We further show that the presence of cyclophilin largely influences the measured ATP hydrolyzing activity of AvaccA in a way that is negatively regulated by the PPIase activity. Taken together, our data support a novel role for cyclophilin in regulating biotin carboxylase activity.

Biochemical characterization of two Azotobacter vinelandii FKBPs and analysis of their interaction with the small subunit of carbamoyl phosphate synthetase.

FK-506 binding proteins (FKBPs) belong to the peptidyl-prolyl cis/trans isomerase superfamily (PPIases, EC: 5.2.1.8) which catalyzes the interconversion of peptidyl-prolyl bonds while they can also act on polypeptides, as folding helper enzymes. Here, we biochemically characterize two recombinant FKBPs, AvfkbA1 and AvfkbA2, from the soil nitrogen-fixing bacterium Azotobacter vinelandii and show that both possess PPIase activity while AvfkbA2 possesses chaperone activity as well. Further, we demonstrate their physical interaction with AvcarA, the small subunit of carbamoyl phosphate synthetase. Using RT-qPCR, we show that AvfkbA1 and AvfkbA2 are co-expressed with AvcarA under the same growth conditions. A decrease in AvfkbA1 or AvfkbA2 PPIase activity, in the presence of AvcarA, further confirms each interaction. However, PPIase activity does not seem to be essential for these interactions since PPIase active site mutations of both FKBPs do not abolish the AvcarA binding. The P(358) residue of AvcarA, possibly retaining a cis configuration, is critical only for the interaction with AvfkbA1. The presence of either of the two FKBPs did not influence the measured glutamine hydrolyzing activity of AvcarA. Taken together, these data indicate that although the two FKBPs have a common biological substrate they probably have differing physiological roles.

The cytoplasmic cyclophilin from Azotobacter vinelandii interacts with phosphate acetyltransferase isoforms enhancing their in vitro activity.

Cyclophilins belong to the peptidyl-prolyl cis/trans isomerase family of enzymes (EC 5.2.1.8), which accelerate protein folding by catalysing the cis/trans isomerisation of proline imidic peptide bonds. In the present study, by a combination of bioinformatics methods, we identify phosphate acetyltransferase isoforms, AvPTA-1 and AvPTA-2, as potential interacting partners of AvPPIB, the cytoplasmic cyclophilin from Azotobacter vinelandii, and demonstrate their physical interaction by co-expression studies. A decrease in AvPPIB PPIase activity, in the presence of AvPTA-1 or AvPTA-2, further confirms each interaction. Phosphate acetyltransferases (EC 2.3.1.8) catalyse the reversible transfer of the acetyl group from acetyl-P to CoA, forming acetyl-CoA and inorganic phosphate. We examined the effect of AvPPIB on the enzymatic activity of both phosphate acetyltransferase isoforms, and noticed an enhancement of the activity, as well as an alteration of the K ( m ) of each isoform, for the reaction substrates, indicating a possible function of AvPPIB in phosphate acetyltransferase activity modulation. Although PPIase activity seems not to be essential for these interactions, since AvPPIB(F99A) active site mutant still interacts with both isoforms, it is responsible for the observed phosphate acetyltransferase activity enhancement as AvPPIB(F99A) enhanced to a significantly lower extent the phosphate acetyltransferase activity of both isoforms compared with AvPPIB.

Characterization of nitrogen-fixing bacteria isolated from field-grown barley, oat, and wheat.

Diazotrophic bacteria were isolated from the rhizosphere of field-grown Triticum aestivum, Hordeum vulgare, and Avena sativa grown in various regions of Greece. One isolate, with the highest nitrogen-fixation ability from each of the eleven rhizospheres, was selected for further characterisation. Diazotrophic strains were assessed for plant-growth-promoting traits such as indoleacetic acid production and phosphate solubilisation. The phylogenies of 16S rRNA gene of the selected isolates were compared with those based on dnaK and nifH genes. The constructed trees indicated that the isolates were members of the species Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. Furthermore, the ipdC gene was detected in all A. brasilence and one A. zeae isolates. The work presented here provides the first molecular genetic evidence for the presence of culturable nitrogen-fixing P. stutzeri and A. zeae associated with field-grown A. sativa and H. vulgare in Greece.

Transcriptional and biochemical characterization of two Azotobacter vinelandii FKBP family members.

Peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), a class of enzymes that catalyse the rate-limiting step of the cis/trans isomerization in protein folding, are divided into three structurally unrelated families: cyclophilins, FK506-binding proteins (FKBPs), and parvulins. Two recombinant FKBPs from the soil nitrogen-fixing bacterium Azotobacter vinelandii, designated as AvfkbX and AvfkbB, have been purified and their peptidyl-prolyl cis/trans isomerase activity against Suc-Ala-Xaa-Pro-Phe-pNA synthetic peptides characterised. The substrate specificity of both enzymes is typical for bacterial FKBPs, with Suc-Ala-Phe-Pro-Phe-pNA being the most rapidly catalysed substrate by AvfkbX and Suc-Ala-Leu-Pro-Phe-pNA by AvfkbB. Both FKBPs display chaperone activity as well in the citrate synthase thermal aggregation assay. Furthermore, using real-time RT-qPCR, we demonstrated that both genes were expressed during the exponential growth phase on glucose minimal medium, while their expression declined dramatically during the stationary growth phase as well as when the growth medium was supplied exogenously with ammonium.