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.

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.

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 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.

Cessation of photosynthesis in Lotus japonicus leaves leads to reprogramming of nodule metabolism.

Symbiotic nitrogen fixation (SNF) involves global changes in gene expression and metabolite accumulation in both rhizobia and the host plant. In order to study the metabolic changes mediated by leaf-root interaction, photosynthesis was limited in leaves by exposure of plants to darkness, and subsequently gene expression was profiled by real-time reverse transcription-PCR (RT-PCR) and metabolite levels by gas chromatography-mass spectrometry in the nodules of the model legume Lotus japonicus. Photosynthetic carbon deficiency caused by prolonged darkness affected many metabolic processes in L. japonicus nodules. Most of the metabolic genes analysed were down-regulated during the extended dark period. In addition to that, the levels of most metabolites decreased or remained unaltered, although accumulation of amino acids was observed. Reduced glycolysis and carbon fixation resulted in lower organic acid levels, especially of malate, the primary source of carbon for bacteroid metabolism and SNF. The high amino acid concentrations together with a reduction in total protein concentration indicate possible protein degradation in nodules under these conditions. Interestingly, comparisons between amino acid and protein content in various organs indicated systemic changes in response to prolonged darkness between nodulated and non-nodulated plants, rendering the nodule a source organ for both C and N under these conditions.

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.

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.

Characterization of two novel nodule-enhanced α-type carbonic anhydrases from Lotus japonicus.

Two cDNA clones coding for α-type carbonic anhydrases (CA; EC 4.2.1.1) in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the full-length proteins was confirmed by heterologous expression in Escherichia coli and purification of the encoded polypeptides. The developmental expression pattern of LjCAA1 and LjCAA2 revealed that both genes code for nodule enhanced carbonic anhydrase isoforms, which are induced early during nodule development. The genes were slightly to moderately down-regulated in ineffective nodules formed by mutant Mesorhizobium loti strains, indicating that these genes may also be involved in biochemical and physiological processes not directly linked to nitrogen fixation/assimilation. The spatial expression profiling revealed that both genes were expressed in nodule inner cortical cells, vascular bundles and central tissue. These results are discussed in the context of the possible roles of CA in nodule carbon dioxide (CO(2)) metabolism.

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.

The Caenorhabditis elegans parvulin gene subfamily and their expression under cold or heat stress along with the fkb subfamily.

Parvulins and FKBPs are members of the peptidyl-prolyl cis/trans isomerases (PPIase) enzyme family whose role is to catalyze the interconversion between the cis trans forms of a peptide bond preceding internal proline residues in a polypeptide substrate. Members of the parvulin subfamily have been found to be involved in a variety of diseases, including Alzheimer's disease and cancer and are also considered possible antiparasitic targets. Genes Y110A2AL.13 (pin-1) and Y48C3A.16 (pin-4) were found in the worm's genome, possibly encoding parvulins. One is homologous to human and fly PIN1 whereas the other is homologous to human and fly PIN4. Both were expressed in Escherichia coli, purified and found to have in vitro PPIase activity. Expression levels of both genes, as well as the fkb genes (that encode FK506-binding proteins) were measured during development and under cold or heat stress conditions. The results revealed a potential role for these genes under temperature-related stress. RNAi silencing was performed for wild type and mutant strain worms under normal and cold or heat stress conditions. A reduced lifespan was observed when pin-4 dsRNA was fed to the fkb-5 deficient worms. Our work presents a first attempt to characterize the Caenorhabditis elegans parvulins and may present an interesting starting point for further experimentation concerning their role, along with the FKBP subfamily, in nematode physiology and their possible use as antiparasitic targets.

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.

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.

Structure of a bacterial cytoplasmic cyclophilin A in complex with a tetrapeptide.

Cyclophilins constitute a class of peptidyl-prolyl isomerases which participate in processes related to protein folding, signalling and chaperoning. The crystal structure of the cytoplasmic cyclophilin A (CyPA) from the bacterium Azotobacter vinelandii complexed with a synthetic tetrapeptide was determined by molecular replacement at 2 resolution. The proline in the tetrapeptide is observed to adopt the cis-isomer conformation. Comparisons of this structure with other CyPA structures provide insights into the conformational variability, effects of peptide binding and structure-function relationships of this enzyme.

Cloning and functional characterization of LjPLT4, a plasma membrane xylitol H(+)- symporter from Lotus japonicus.

Polyols are compounds that play various physiological roles in plants. Here we present the identification of four cDNA clones of the model legume Lotus japonicus, encoding proteins of the monosaccharide transporter-like (MST) superfamily that share significant homology with previously characterized polyol transporters (PLTs). One of the transporters, named LjPLT4, was characterized functionally after expression in yeast. Transport assays revealed that LjPLT4 is a xylitol-specific H(+)-symporter (K (m), 0.34 mM). In contrast to the previously characterized homologues, LjPLT4 was unable to transport other polyols, including mannitol, sorbitol, myo-inositol and galactitol, or any of the monosaccharides tested. Interestingly, some monosaccharides, including fructose and xylose, inhibited xylitol uptake, although no significant uptake of these compounds was detected in the LjPLT4 transformed yeast cells, suggesting interactions with the xylitol binding site. Subcellular localization of LjPLT4-eYFP fusions expressed in Arabidopsis leaf epidermal cells indicated that LjPLT4 is localized in the plasma membrane. Real-time RT-PCR revealed that LjPLT4 is expressed in all major plant organs, with maximum transcript accumulation in leaves correlating with maximum xylitol levels there, as determined by GC-MS. Thus, LjPLT4 is the first plasma membrane xylitol-specific H(+)-symporter to be characterized in plants.

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.

Nodulation enhances dark CO2 fixation and recycling in the model legume Lotus japonicus.

During symbiotic nitrogen fixation (SNF), the nodule becomes a strong sink for photosynthetic carbon. Here, it was studied whether nodule dark CO(2) fixation could participate in a mechanism for CO(2) recycling through C(4)-type photosynthesis. Differences in the natural δ(13)C abundance between Lotus japonicus inoculated or not with the N-fixing Mesorhizobium loti were assessed. (13)C labelling and gene expression of key enzymes of CO(2) metabolism were applied in plants inoculated with wild-type or mutant fix(-) (deficient in N fixation) strains of M. loti, and in non-inoculated plants. Compared with non-inoculated legumes, inoculated legumes had higher natural δ(13)C abundance and total C in their hypergeous organs and nodules. In stems, (13)C accumulation and expression of genes coding for enzymes of malate metabolism were greater in inoculated compared with non-inoculated plants. Malate-oxidizing activity was localized in stem xylem parenchyma, sieve tubes, and photosynthetic outer cortex parenchyma of inoculated plants. In stems of plants inoculated with fix(-) M. loti strains, (13)C accumulation remained high, while accumulation of transcripts coding for malic enzyme isoforms increased. A potential mechanism is proposed for reducing carbon losses during SNF by the direct reincorporation of CO(2) respired by nodules and the transport and metabolism of C-containing metabolites in hypergeous organs.

The genetic diversity of culturable nitrogen-fixing bacteria in the rhizosphere of wheat.

A total of 17 culturable nitrogen-fixing bacterial strains associated with the roots of wheat growing in different regions of Greece were isolated and characterized for plant-growth-promoting traits such as auxin production and phosphate solubilization. The phylogenetic position of the isolates was first assessed by the analysis of the PCR-amplified 16S rRNA gene. The comparative sequence analysis and phylogenetic analysis based on 16S rRNA gene sequences show that the isolates recovered in this study are grouped with Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. The diazotrophic nature of all isolates was confirmed by amplification of partial nifH gene sequences. The phylogenetic tree based on nifH gene sequences is consistent with 16S rRNA gene phylogeny. The isolates belonging to Azospirillum species were further characterized by examining the partial dnaK gene phylogenetic tree. Furthermore, it was demonstrated that the ipdC gene was present in all Azospirillum isolates, suggesting that auxin is mainly synthesized via the indole-3-pyruvate pathway. Although members of P. stutzeri and A. zeae are known diazotrophic bacteria, to the best of our knowledge, this is the first report of isolation and characterization of strains belonging to these bacterial genera associated with wheat.

Cloning, characterization and transcriptional analysis of two phosphate acetyltransferase isoforms from Azotobacter vinelandii.

Acetate is abundant in soil contributing to a great extent on carbon cycling in nature. Phosphate acetyltransferase (Pta, EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl-P to CoA forming acetyl-CoA and inorganic phosphate, participating to acetate assimilation/dissimilation reactions. In the present study, we demonstrate that Azotobacter vinelandii, a nitrogen-fixing, free-living, soil bacterium, possesses two class II phosphate acetyltransferase isoforms, AvPTA-1 and AvPTA-2, with different kinetic properties. At the acetyl-CoA forming direction, AvPTA-1 has lower affinity for acetyl-P and higher affinity for CoA than AvPTA-2 while at the acetyl-P forming direction; activity was measured only for AvPTA-1. Quantification of their expression patterns by RT-qPCR indicated that both genes are expressed during exponential growth on glucose or acetate and are down-regulated in the stationary phase. The ammonium availability during acetate growth resulted in up-regulation of Avpta-2 expression only. Further, the gene expression patterns of other related gene transcripts were also investigated, in order to understand the influence of each pathway in the assimilation/dissimilation of acetate.

Molecular and biochemical analysis of the α class carbonic anhydrases in Caenorhabditis elegans.

In this study, in silico analysis of the Caenorhabditis elegans genome revealed six genes (cah-1, cah-2, cah-3, cah-4, cah-5, and cah-6) possibly encoding α class CAs (carbonic anhydrase). Real-time RT-PCR analysis revealed the temporal expression pattern of each gene, as well as changes in expression levels under different atmospheric conditions (stress). Cah-3 and cah-4 showed the highest levels of transcript accumulation, while most genes responded to the stress conditions. Yeast complementation showed that cah-3 was able to complement the function of Saccharomyces cerevisiae CA (NCE103) in vivo. Recombinant CAH-3, CAH-4a and CAH-5 enzymes, expressed in Escherichia coli were used for in vitro measurement of CA activity. However, in vitro activity was only detectable for CAH-4a. RNAi by feeding was performed on wild-type C. elegans for all genes. The worms were examined for a visible phenotype under normal and stress conditions (pH, CO(2)/O(2)). Silencing cah-3 and cah-4 may reduce the life-span of the worms (at 22 °C).