Roles of quorum sensing molecules from Rhizobium etli RT1 in bacterial motility and biofilm formation

ABSTRACT Strain RT1 was isolated from root nodules of Lens culinaris (a lentil) and characterized as Rhizobium etli (a Gram-negative soil-borne bacterium) by 16S rDNA sequencing and phylogenetic analysis. The signaling molecules produced by R. etli (RT1) were detected and identified by high-performance liquid chromatography coupled with mass spectrometry. The most abundant and biologically active N-acyl homoserine lactone molecules (3-oxo-C8-HSL and 3-OH-C14-HSL) were detected in the ethyl acetate extract of RT1. The biological role of 3-oxo-C8-HSL was evaluated in RT1. Bacterial motility and biofilm formation were affected or modified on increasing concentrations of 3-oxo-C8-HSL. Results confirmed the existence of cell communication in RT1 mediated by 3-oxo-C8-HSL, and positive correlations were found among quorum sensing, motility and biofilm formation in RT1.

Transcriptional Activation of Virulence Genes of Rhizobium etli.

Recently, Rhizobium etli, in addition to Agrobacterium spp., has emerged as a prokaryotic species whose genome encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we show that the signal phenolic molecule acetosyringone (AS) induces R. etli vir gene expression both in an R. etli background and in an Agrobacterium tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter transfer DNA (T-DNA). Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium but not from nonhost plants. The early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a than of T-DNA transfer of pTiC58 of A. tumefaciensIMPORTANCE The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by the genome of a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into the plant cell genome. In this study, we explored the transcriptional regulation and induction of virulence genes in R. etli and show similarities to and differences from those of their A. tumefaciens counterparts, contributing to an understanding and a comparison of these two systems. Whereas most vir genes in R. etli follow an induction pattern similar to that of A. tumefaciens vir genes, a few significant differences may at least in part explain the variations in T-DNA transfer efficiency.

Quinol oxidase encoded by cyoABCD in Rhizobium etli CFN42 is regulated by ActSR and is crucial for growth at low pH or low iron conditions.

Rhizobium etli aerobically respires with several terminal oxidases. The quinol oxidase (Cyo) encoded by cyoABCD is needed for efficient adaptation to low oxygen conditions and cyo transcription is upregulated at low oxygen. This study sought to determine how transcription of the cyo operon is regulated. The 5' sequence upstream of cyo was analysed in silico and revealed putative binding sites for ActR of the ActSR two-component regulatory system. The expression of cyo was decreased in an actSR mutant regardless of the oxygen condition. As ActSR is known to be important for growth under low pH in another rhizobial species, the effect of growth medium pH on cyo expression was tested. As the pH of the media was incrementally decreased, cyo expression gradually increased in the WT, eventually reaching ∼ 10-fold higher levels at low pH (4.8) compared with neutral pH (7.0) conditions. This upregulation of cyo under decreasing pH conditions was eliminated in the actSR mutant. Both the actSR and cyo mutants had severe growth defects at low pH (4.8). Lastly, the actSR and cyo mutants had severe growth defects when grown in media treated with an iron chelator. Under these conditions, cyo was upregulated in the WT, whereas cyo was not induced in the actSR mutant. Altogether, the results indicated cyo expression is largely dependent on the ActSR two-component system. This study also demonstrated additional physiological roles for Cyo in R. etli CFN42, in which it is the preferred oxidase for growth under acidic and low iron conditions.

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.

RepA and RepB exert plasmid incompatibility repressing the transcription of the repABC operon.

Rhizobium etli CFN42 has a multipartite genome composed of one chromosome and six large plasmids with low copy numbers, all belonging to the repABC plasmid family. All elements essential for replication and segregation of these plasmids are encoded within the repABC operon. RepA and RepB direct plasmid segregation and are involved in the transcriptional regulation of the operon, and RepC is the initiator protein of the plasmid. Here we show that in addition to RepA (repressor) and RepB (corepressor), full transcriptional repression of the operon located in the symbiotic plasmid (pRetCFN42d) of this strain requires parS, the centromere-like sequence, and the operator sequence. However, the co-expression of RepA and RepB is sufficient to induce the displacement of the parental plasmid. RepA is a Walker-type ATPase that self associates in vivo and in vitro and binds specifically to the operator region in its RepA-ADP form. In contrast, RepA-ATP is capable of binding to non-specific DNA. RepA and RepB form high molecular weight DNA-protein complexes in the presence of ATP and ADP. RepA carrying ATP-pocket motif mutations induce full repression of the repABC operon without the participation of RepB and parS. These mutants specifically bind the operator sequence in their ATP or ADP bound forms. In addition, their expression in trans exerts plasmid incompatibility against the parental plasmid. RepA and RepB expressed in trans induce plasmid incompatibility because of their ability to repress the repABC operon and not only by their capacity to distort the plasmid segregation process.

Rhizobium etli asparaginase II: an alternative for acute lymphoblastic leukemia (ALL) treatment.

Bacterial L-asparaginase has been a universal component of therapies for childhood acute lymphoblastic leukemia since the 1970s. Two principal enzymes derived from Escherichia coli and Erwinia chrysanthemi are the only options clinically approved to date. We recently reported a study of recombinant L-asparaginase (AnsA) from Rhizobium etli and described an increasing type of AnsA family members. Sequence analysis revealed four conserved motifs with notable differences with respect to the conserved regions of amino acid sequences of type I and type II L-asparaginases, particularly in comparison with therapeutic enzymes from E. coli and E. chrysanthemi. These differences suggested a distinct immunological specificity. Here, we report an in silico analysis that revealed immunogenic determinants of AnsA. Also, we used an extensive approach to compare the crystal structures of E. coli and E. chrysantemi asparaginases with a computational model of AnsA and identified immunogenic epitopes. A three-dimensional model of AsnA revealed, as expected based on sequence dissimilarities, completely different folding and different immunogenic epitopes. This approach could be very useful in transcending the problem of immunogenicity in two major ways: by chemical modifications of epitopes to reduce drug immunogenicity, and by site-directed mutagenesis of amino acid residues to diminish immunogenicity without reduction of enzymatic activity.

Glutathione is required by Rhizobium etli for glutamine utilization and symbiotic effectiveness.

Here, we provide genetic and biochemical evidence indicating that the ability of Rhizobium etli bacteria to efficiently catabolize glutamine depends on its ability to produce reduced glutathione (l-γ-glutamyl-l-cysteinylglycine [GSH]). We find that GSH-deficient strains, namely a gshB (GSH synthetase) and a gor (GSH reductase) mutant, can use different amino acids, including histidine, alanine, and asparagine but not glutamine, as sole source of carbon, energy, and nitrogen. Moreover, l-buthionine(S,R)-sulfoximine, a GSH synthesis inhibitor, or diamide that oxidizes GSH, induced the same phenotype in the wild-type strain. Among the steps required for its utilization, glutamine uptake, occurring through the two well-characterized carriers (Aap and Bra systems) but not glutamine degradation or respiration, was largely reduced in GSH-deficient strains. Furthermore, GSH-deficient mutants of R. etli showed a reduced symbiotic efficiency. Exogenous GSH was sufficient to rescue glutamine uptake or degradation ability, as well as the symbiotic effectiveness of GSH mutants. Our results suggest a previously unknown GSH-glutamine metabolic relationship in bacteria.

Novel insights into the biotin carboxylase domain reactions of pyruvate carboxylase from Rhizobium etli.

The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO(3)(-) deprotonation (Glu305 and Arg301), and biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO(3)(-), Lys245, Glu218, and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the biotin carboxylase and carboxyl transferase domains were uncoupled in the R353M-catalyzed reactions, indicating that Arg353 may not only facilitate the formation of the biotin enolate but also assist in coordinating catalysis between the two spatially distinct active sites. The 2.5- and 4-fold increase in k(cat) for the full reverse reaction with the R353K and R353M mutants, respectively, suggests that mutation of Arg353 allows carboxybiotin increased access to the biotin carboxylase domain active site. The proposed chemical mechanism is initiated by the deprotonation of HCO(3)(-) by Glu305 and concurrent nucleophilic attack on the γ-phosphate of MgATP. The trianionic carboxyphosphate intermediate formed reversibly decomposes in the active site to CO(2) and PO(4)(3-). PO(4)(3-) then acts as the base to deprotonate the tethered biotin at the N(1)-position. Stabilized by interactions between the ureido oxygen and Arg353, the biotin-enolate reacts with CO(2) to give carboxybiotin. The formation of a distinct salt bridge between Arg353 and Glu248 is proposed to aid in partially precluding carboxybiotin from reentering the biotin carboxylase active site, thus preventing its premature decarboxylation prior to the binding of a carboxyl acceptor in the carboxyl transferase domain.

Activation and inhibition of pyruvate carboxylase from Rhizobium etli.

While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg(2+), and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg(2+) assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg(2+) vs pyruvate were nonlinear at low concentrations of Mg(2+) and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg(2+) also resulted in a decrease in the K(a) for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K(i) = 0.026 mM) and pyruvate carboxylation (K(i) = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg(2+) metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg(2+) are described.

Cloning and characterization of a thermostable xylitol dehydrogenase from Rhizobium etli CFN42.

An NAD(+)-dependent xylitol dehydrogenase from Rhizobium etli CFN42 (ReXDH) was cloned and overexpressed in Escherichia coli. The DNA sequence analysis revealed an open reading frame of 1,044 bp, capable of encoding a polypeptide of 347 amino acid residues with a calculated molecular mass of 35,858 Da. The ReXDH protein was purified as an active soluble form using GST affinity chromatography. The molecular mass of the purified enzyme was estimated to be approximately 34 kDa by sodium dodecyl sulfate-polyacrylamide gel and approximately 135 kDa with gel filtration chromatography, suggesting that the enzyme is a homotetramer. Among various polyols, xylitol was the preferred substrate of ReXDH with a K (m) = 17.9 mM and k(cat) /K (m) = 0.5 mM(-1) s(-1) for xylitol. The enzyme had an optimal pH and temperature of 9.5 and 70 degrees C, respectively. Heat inactivation studies revealed a half life of the ReXDH at 40 degrees C of 120 min and a half denaturation temperature (T (1/2)) of 53.1 degrees C. ReXDH showed the highest optimum temperature and thermal stability among the known XDHs. Homology modeling and sequence analysis of ReXDH shed light on the factors contributing to the high thermostability of ReXDH. Although XDHs have been characterized from several other sources, ReXDH is distinguished from other XDHs by its high thermostability.

Pleiotropic effects of a rel mutation on stress survival of Rhizobium etli CNPAF512.

BACKGROUND: The rel gene of Rhizobium etli (relRet), the nodulating endosymbiont of the common bean plant, determines the cellular level of the alarmone (p)ppGpp and was previously shown to affect free-living growth and symbiosis. Here, we demonstrate its role in cellular adaptation and survival in response to various stresses. RESULTS: Growth of the R. etli relRet mutant was strongly reduced or abolished in the presence of elevated NaCl levels or at 37 degrees C, compared to the wild type. In addition, depending on the cell density, decreased survival of exponentially growing or stationary phase relRet mutant cells was obtained after H2O2, heat or NaCl shock compared to the wild-type strain. Survival of unstressed stationary phase cultures was differentially affected depending on the growth medium used. Colony forming units (CFU) of relRet mutant cultures continuously decreased in minimal medium supplemented with succinate, whereas wild-type cultures stabilised at higher CFU levels. Microscopic examination of stationary phase cells indicated that the relRet mutant was unable to reach the typical coccoid morphology of the wild type in stationary phase cultures. Assessment of stress resistance of re-isolated bacteroids showed increased sensitivity of the relRet mutant to H2O2 and a slightly increased resistance to elevated temperature (45 degrees C) or NaCl shock, compared to wild-type bacteroids. CONCLUSION: The relRet gene is an important factor in regulating rhizobial physiology, during free-living growth as well as in symbiotic conditions. Additionally, differential responses to several stresses applied to bacteroids and free-living exponential or stationary phase cells point to essential physiological differences between the different states.

Requirement of a plasmid-encoded catalase for survival of Rhizobium etli CFN42 in a polyphenol-rich environment.

Nitrogen-fixing bacteria collectively called rhizobia are adapted to live in polyphenol-rich environments. The mechanisms that allow these bacteria to overcome toxic concentrations of plant polyphenols have not been clearly elucidated. We used a crude extract of polyphenols released from the seed coat of the black bean to simulate a polyphenol-rich environment and analyze the response of the bean-nodulating strain Rhizobium etli CFN42. Our results showed that the viability of the wild type as well as that of derivative strains cured of plasmids p42a, p42b, p42c, and p42d or lacking 200 kb of plasmid p42e was not affected in this environment. In contrast, survival of the mutant lacking plasmid p42f was severely diminished. Complementation analysis revealed that the katG gene located on this plasmid, encoding the only catalase present in this bacterium, restored full resistance to testa polyphenols. Our results indicate that oxidation of polyphenols due to interaction with bacterial cells results in the production of a high quantity of H(2)O(2), whose removal by the katG-encoded catalase plays a key role for cell survival in a polyphenol-rich environment.

The Rhizobium etli bioMNY operon is involved in biotin transport.

Because Rhizobium etli CE3 is normally dependent on an external source of biotin and lacks orthodox biotin biosynthesis genes, we undertook an analysis of biotin uptake in this organism. By complementation of a Sinorhizobium meliloti bioM mutant we isolated an R. etli chromosomal region encoding homologs of the S. meliloti bioMNB genes, whose products have been implicated in intracellular biotin retention in that organism. Disruption of the R. etli bioM resulted in a mutant which took up biotin at a lower rate and accumulated significantly less biotin than the wild type. As in S. meliloti, the R. etli bioMN gene-products resemble the ATPase and permease components, respectively, of an ABC-type transporter. The bioB gene product is in fact similar to members of the BioY family, which has been postulated to function in biotin transport, and we refer to this gene as bioY. An R. etli bioY mutant exhibited lower biotin uptake than the wild-type, providing the first experimental evidence for a role of BioY in biotin transport. We show that the bioMNY operon is transcriptionally repressed by biotin. An analysis of the competitiveness of the wild-type strain versus the bioM mutant showed that the mutant had a diminished capacity to form nodules on bean plants.

Effective symbiosis between Rhizobium etli and Phaseolus vulgaris requires the alarmone ppGpp.

The symbiotic interaction between Rhizobium etli and Phaseolus vulgaris, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The R. etli relA gene was identified through a genome-wide screening for R. etli symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an R. etli relA mutant strain under conditions of amino acid starvation. Plants nodulated by an R. etli relA mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of relA mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the sigma(N)-dependent nitrogen fixation genes rpoN2 and iscN was considerably reduced in the relA mutant. In addition, the expression of the relA gene was negatively regulated by RpoN2, the symbiosis-specific sigma(N) copy of R. etli. Therefore, an autoregulatory loop controlling the expression of relA and rpoN2 seems operative in bacteroids. The production of long- and short-chain acyl-homoserine-lactones by the cinIR and raiIR systems was decreased in an R. etli relA mutant. Our results suggest that relA may play an important role in the regulation of gene expression in R. etli bacteroids and in the adaptation of bacteroid physiology.

Three genes encoding for putative methyl- and acetyltransferases map adjacent to the wzm and wzt genes and are essential for O-antigen biosynthesis in Rhizobium etli CE3.

The elucidation of the structure of the O-antigen of Rhizobium etli CE3 predicts that the R. etli CE3 genome must contain genes encoding acetyl- and methyltransferases to confer the corresponding modifications to the O-antigen. We identified three open reading frames (ORFs) upstream of wzm, encoding the membrane component of the O-antigen transporter and located in the lps alpha-region of R. etli CE3. The ORFs encode two putative acetyltransferases with similarity to the CysE-LacA-LpxA-NodL family of acetyltransferases and one putative methyltransferase with sequence motifs common to a wide range of S-adenosyl-L-methionine-dependent methyltransferases. Mutational analysis of the ORFs encoding the putative acetyltransferases and methyltransferase revealed that the acetyl and methyl decorations mediated by these specific enzymes are essential for O-antigen synthesis. Composition analysis and high performance anion exchange chromatography analysis of the lipopolysaccharides (LPSs) of the mutants show that all of these LPSs contain an intact core region and lack the O-antigen polysaccharide. The possible role of these transferases in the decoration of the O-antigen of R. etli is discussed.