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The LysR-type transcriptional regulators (LTTRs) are DNA-binding proteins present in bacteria, archaea, and in algae. Knowledge about their distribution, abundance, evolution, structural organization, transcriptional regulation, fundamental roles in free life, pathogenesis, and bacteria–plant interaction has been generated. This review focuses on these aspects and provides a current picture of LTTR biology. [ABSTRACT FROM AUTHOR]

A characterization of the LtrR regulator, an S. Typhi protein belonging to the LysR family is presented. Proteomics, outer membrane protein profiles and transcriptional analyses demonstrated that LtrR is required for the synthesis of OmpR, OmpC and OmpF. DNA-protein interaction analysis showed that LtrR binds to the regulatory region of ompR and then OmpR interacts with the ompC and ompF promoters inducing porin synthesis. LtrR-dependent and independent ompR promoters were identified, and both promoters are involved in the synthesis of OmpR for OmpC and OmpF production. To define the functional role of the ltrR- ompR- ompC- ompF genetic network, mutants in each gene were obtained. We found that ltrR, ompR, ompC and ompF were involved in the control of bacterial transformation, while the two regulators and ompC are necessary for the optimal growth of S. Typhi in the presence of one of the major bile salts found in the gut, sodium deoxycholate. The data presented establish the pivotal role of LtrR in the regulatory network of porin synthesis and reveal new genetic strategies of survival and cellular adaptation to the environment used by S almonella. [ABSTRACT FROM AUTHOR]

LtrR is a LysR-type regulator involved in the positive expression of ompR to promote ompC and ompF expression. This regulatory network is fundamental for the control of bacterial transformation and resistance to the bile salt sodium deoxycholate in Salmonella enterica serovar Typhi. In this work, the transcriptional regulation of ltrR was characterized, revealing that the use of alternative promoters results in two transcripts. The larger one, the ltrR2 mRNA, was repressed at promoter and coding regions by H-NS, whereas Lrp repressed its expression at the coding region. In the case of the second and shorter ltrR1 transcript, it was repressed only at the coding region by H-NS and Lrp. Remarkably, pH 7.5 is a positive signal involved in the transcriptional expression of both ltrR units. Translational fusions and Western blot experiments demonstrated that ltrR2 and ltrR1 mRNAs encode the LtrR2 and LtrR1 proteins. This study adds new data on the complex genetic and regulatory characteristics of one of the most predominant types of transcriptional factors in bacteria, the LysR-type transcriptional regulators. [ABSTRACT FROM AUTHOR]

Introduction. Salmonella enterica serovar Typhi ( S . Typhi) is the etiological agent of typhoid fever. To establish an infection in the human host, this pathogen must survive the presence of bile salts in the gut and gallbladder. Hypothesis. S . Typhi uses multiple genetic elements to resist the presence of human bile. Aims. To determine the genetic elements that S . Typhi utilizes to tolerate the human bile salt sodium deoxycholate. Methodology. A collection of S . Typhi mutant strains was evaluated for their ability to growth in the presence of sodium deoxycholate and ox-bile. Additionally, transcriptomic and proteomic responses elicited by sodium deoxycholate on S . Typhi cultures were also analysed. Results. Multiple transcriptional factors and some of their dependent genes involved in central metabolism, as well as in cell envelope, are required for deoxycholate resistance. Conclusion. These findings suggest that metabolic adaptation to bile is focused on enhancing energy production to sustain synthesis of cell envelope components exposed to damage by bile salts.

The CRISPR-Cas cluster is found in many prokaryotic genomes including those of the Enterobacteriaceae family. Salmonella enterica serovar Typhi ( S . Typhi) harbors a Type I-E CRISPR-Cas locus composed of cas3 , cse1 , cse2 , cas7 , cas5 , cas6e , cas1 , cas2 , and a CRISPR1 array. In this work, it was determined that, in the absence of cas5 or cas2 , the amount of the OmpC porin decreased substantially, whereas in individual cse2 , cas6e , cas1 , or cas3 null mutants, the OmpF porin was not observed in an electrophoretic profile of outer membrane proteins. Furthermore, the LysR-type transcriptional regulator LeuO was unable to positively regulate the expression of the quiescent OmpS2 porin, in individual S . Typhi cse2 , cas5 , cas6e , cas1 , cas2 , and cas3 mutants. Remarkably, the expression of the master porin regulator OmpR was dependent on the Cse2, Cas5, Cas6e, Cas1, Cas2, and Cas3 proteins. Therefore, the data suggest that the CRISPR-Cas system acts hierarchically on OmpR to control the synthesis of outer membrane proteins in S . Typhi., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Medina-Aparicio, Rodriguez-Gutierrez, Rebollar-Flores, Martínez-Batallar, Mendoza-Mejía, Aguirre-Partida, Vázquez, Encarnación, Calva and Hernández-Lucas.)

The ability of Escherichia coli to grow on L-lactate as a sole carbon source depends on the expression of the lldPRD operon. A striking feature of this operon is that the transcriptional regulator (LldR) encoding gene is located between the permease (LldP) and the dehydrogenase (LldD) encoding genes. In this study we report that dosage of the LldP, LldR, and LldD proteins is not modulated on the transcriptional level. Instead, modulation of protein dosage is primarily correlated with RNase E-dependent mRNA processing events that take place within the lldR mRNA, leading to the immediate inactivation of lldR , to differential segmental stabilities of the resulting cleavage products, and to differences in the translation efficiencies of the three cistrons. A model for the processing events controlling the molar quantities of the proteins in the lldPRD operon is presented and discussed. Importance Adjustment of gene expression is critical for proper cell function. For the case of polycistronic transcripts, posttranscriptional regulatory mechanisms can be used to fine-tune the expression of individual cistrons. Here, we elucidate how protein dosage of the Escherichia coli lldPRD operon, which presents the paradox of having the gene encoding a regulator protein located between genes that code for a permease and an enzyme, is regulated. Our results demonstrate that the key event in this regulatory mechanism involves the RNase E-dependent cleavage of the primary lldPRD transcript at internal site(s) located within the lldR cistron, resulting in a drastic decrease of intact lldR mRNA, to differential segmental stabilities of the resulting cleavage products, and to differences in the translation efficiencies of the three cistrons., (Copyright © 2020 American Society for Microbiology.)

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 ( smc03091 ) and argI2 ( sma1711 ). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni 2+ as cofactor. The enzyme was also active with Mn 2+ and Co 2+ , both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator ( smc03092 ) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti , that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu 2+ ) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu 2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu 2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu 2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H2O2 concentration, lipoperoxidation and accumulate 40-fold more Cu 2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.

One of the most commonly used tools to compare protein or DNA sequences against databases is BLAST. We introduce a web tool that allows the performance of BLAST-searches of protein/DNA sequences in whole-genome sequenced bacteria/archaea, and displays a large amount of BLAST-results simultaneously. The circular bacterial replicons are projected as horizontal lines with fixed length of 360, representing the degrees of a circle. A coordinate system is created with length of the replicon along the x -axis and the number of replicon used on the y -axis. When a query sequence matches with a gene/protein of a particular replicon, the BLAST-results are depicted as an " x,y " position in a specially adapted plot. This tool allows the visualization of the results from the whole data to a particular gene/protein in real time with low computational resources., (Copyright © 2018 Pedraza-Pérez et al.)

In nature, microorganisms are constantly exposed to multiple viral infections and thus have developed many strategies to survive phage attack and invasion by foreign DNA. One of such strategies is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) bacterial immunological system. This defense mechanism is widespread in prokaryotes including several families such as Enterobacteriaceae. Much knowledge about the CRISPR-Cas system has been generated, including its biological functions, transcriptional regulation, distribution, utility as a molecular marker and as a tool for specific genome editing. This review focuses on these aspects and describes the state of the art of the CRISPR-Cas system in the Enterobacteriaceae bacterial family., (© FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Methanol-consuming culturable bacteria were isolated from the plant surface, rhizosphere, and inside the stem of Neobuxbaumia macrocephala. All 38 isolates were facultative methylotrophic microorganisms. Their classification included the Classes Actinobacteria, Sphingobacteriia, Alpha-, Beta-, and Gammaproteobacteria. The deduced amino acid sequences of methanol dehydrogenase obtained by PCR belonging to Actinobacteria, Alpha-, Beta-, and Gammaproteobacteria showed high similarity to rare-earth element (REE)-dependent XoxF methanol dehydrogenases, particularly the group XoxF5. The sequences included Asp 301 , the REE-coordinating amino acid, present in all known XoxF dehydrogenases and absent in MxaF methanol dehydrogenases. The quantity of the isolates showed positive hybridization with a xoxF probe, but not with a mxaF probe. Isolates of all taxonomic groups showed methylotrophic growth in the presence of Ce 3+ or Ca 2+ . The presence of xoxF-like sequences in methylotrophic bacteria from N. macrocephala and its potential relationship with their adaptability to xerophytic plants are discussed.

The CRISPR-Cas system is involved in bacterial immunity, virulence, gene regulation, biofilm formation and sporulation. In Salmonella enterica serovar Typhi, this system consists of five transcriptional units including antisense RNAs. It was determined that these genetic elements are expressed in minimal medium and are up-regulated by pH. In addition, a transcriptional characterization of cas3 and ascse2-1 is included herein.

L-Ornithine production in the alfalfa microsymbiont Sinorhizobium meliloti occurs as an intermediate step in arginine biosynthesis. Ornithine is required for effective symbiosis but its synthesis in S. meliloti has been little studied. Unlike most bacteria, S. meliloti 1021 is annotated as encoding two enzymes producing ornithine: N-acetylornithine (NAO) deacetylase (ArgE) hydrolyses NAO to acetate and ornithine, and glutamate N-acetyltransferase (ArgJ) transacetylates l-glutamate with the acetyl group from NAO, forming ornithine and N-acetylglutamate (NAG). NAG is the substrate for the second step of arginine biosynthesis catalysed by NAG kinase (ArgB). Inactivation of argB in strain 1021 resulted in arginine auxotrophy. The activity of purified ArgB was significantly inhibited by arginine but not by ornithine. The purified ArgJ was highly active in NAO deacetylation/glutamate transacetylation and was significantly inhibited by ornithine but not by arginine. The purified ArgE protein (with a 6His-Sumo affinity tag) was also active in deacetylating NAO. argE and argJ single mutants, and an argEJ double mutant, are arginine prototrophs. Extracts of the double mutant contained aminoacylase (Ama) activity that deacetylated NAO to form ornithine. The purified products of three candidate ama genes (smc00682 (hipO1), smc02256 (hipO2) and smb21279) all possessed NAO deacetylase activity. hipO1 and hipO2, but not smb21279, expressed in trans functionally complemented an Escherichia coli ΔargE : : Km mutant. We conclude that Ama activity accounts for the arginine prototrophy of the argEJ mutant. Transcriptional assays of argB, argE and argJ, fused to a promoterless gusA gene, showed that their expression was not significantly affected by exogenous arginine or ornithine.

Manganese (Mn(2+)) plays a key role in important cellular functions such as oxidative stress response and bacterial virulence. The mechanisms of Mn(2+) homeostasis are not fully understood, there are few data regarding the functional and taxonomic diversity of Mn(2+) exporters. Our recent phylogeny of the cation diffusion facilitator (CDF) family of transporters classified the bacterial Mn(2+)-CDF transporters characterized to date, Streptococcus pneumoniae MntE and Deinococcus radiodurans DR1236, into two monophyletic groups. DR1236 was shown to belong to the highly-diverse metal specificity clade VI, together with TtCzrB, a Zn(2+)/Cd(2+) transporter from Thermus thermophilus, the Fe(2+) transporter Sll1263 from Synechocystis sp and eight uncharacterized homologs whose potential Mn(2+)/Zn(2+)/Cd(2+)/Fe(2+) specificities could not be accurately inferred because only eleven proteins were grouped in this clade. A new phylogeny inferred from the alignment of 197 clade VI homologs revealed three novel subfamilies of uncharacterized proteins. Remarkably, one of them contained 91 uncharacterized α-proteobacteria transporters (46% of the protein data set) grouped into a single subfamily. The Mn(2+)/Fe(2+) specificity of this subfamily was proposed through the functional characterization of the Rhizobium etli RHE_CH03072 gene. This gene was upregulated by Mn(2+), Zn(2+), Cd(2+) and Fe(2+) but conferred only Mn(2+) resistance to R. etli. The expression of the RHE_CH03072 gene in an E. coli mntP/zitB/zntA mutant did not relieve either Zn(2+) or Mn(2+) stress but slightly increased its Fe(2+) resistance. These results indicate that the RHE_CH03072 gene, now designated as emfA, encodes for a bacterial Mn(2+)/Fe(2+) resistance CDF protein, having orthologs in more than 60 α-proteobacterial species.

LeuO is a LysR-type transcriptional regulator (LTTR) that has been described to be a global regulator in Escherichia coli and Salmonella enterica, since it positively and negatively regulates the expression of genes involved in multiple biological processes. LeuO is comprised of an N-terminal DNA-binding domain (DBD) with a winged helix-turn-helix (wHTH) motif and of a long linker helix (LH) involved in dimerization that connects the DBD with the C-terminal effector-binding domain (EBD) or regulatory domain (RD; which comprises subdomains RD-I and RD-II). Here we show that the oligomeric structure of LeuO is a tetramer that binds with high affinity to DNA. A collection of single amino acid substitutions in the LeuO DBD indicated that this region is involved in oligomerization, in positive and negative regulation, as well as in DNA binding. Mutants with point mutations in the central and C-terminal regions of RD-I were affected in transcriptional activation. Deletion of the RD-II and RD-I C-terminal subdomains affected not only oligomerization but also DNA interaction, showing that they are involved in positive and negative regulation. Together, these data demonstrate that not only the C terminus but also the DBD of LeuO is involved in oligomer formation; therefore, each LeuO domain appears to act synergistically to maintain its regulatory functions in Salmonella enterica serovar Typhi., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

LeuO is a quiescent genetic regulator present in many bacteria, which forms part of the H-NS regulon. LeuO in turn has been proposed to activate a subset of genes of the regulon by antagonizing H-NS. In the paper by Dillon et al., binding of LeuO to the S. Typhimurium genome was observed by ChIP-chip to some of the previously described LeuO-regulated genes, upon growth under stress conditions. However, studies at a higher LeuO concentration from a cloned inducible promoter rendered many more binding sites, pointing towards the importance of the abundance of the regulator in the cell, in a given moment. Binding of LeuO was observed not only to intergenic sequences, but in the majority of cases to intragenic sequences, and co-binding was observed with H-NS in many sites and with RNA polymerase to the majority of sites. The authors define a binding motif that allowed the detection of several other LeuO-regulated genes that were not detected by ChIP-chip, which were possibly missed because LeuO binds and bridges distal sites, in those instances. The observations reported open new questions regarding the mode of action for LeuO., (© 2012 Blackwell Publishing Ltd.)

The genus Burkholderia includes pathogens of plants and animals and some human opportunistic pathogens, such as the Burkholderia cepacia complex (Bcc), but most species are nonpathogenic, plant associated, and rhizospheric or endophytic. Since rhizobacteria expressing ACC (1-aminocyclopropane-1-carboxylate) deaminase may enhance plant growth by lowering plant ethylene levels, in this work we investigated the presence of ACC deaminase activity and the acdS gene in 45 strains, most of which are plant associated, representing 20 well-known Burkholderia species. The results demonstrated that ACC deaminase activity is a widespread feature in the genus Burkholderia, since 18 species exhibited ACC deaminase activities in the range from 2 to 15 mumol of alpha-ketobutyrate/h/mg protein, which suggests that these species may be able to modulate ethylene levels and enhance plant growth. In these 18 Burkholderia species the acdS gene sequences were highly conserved (76 to 99% identity). Phylogenetic analysis of acdS gene sequences in Burkholderia showed tight clustering of the Bcc species, which were clearly distinct from diazotrophic plant-associated Burkholderia species. In addition, an acdS knockout mutant of the N(2)-fixing bacterium Burkholderia unamae MTl-641(T) and a transcriptional acdSp-gusA fusion constructed in this strain showed that ACC deaminase could play an important role in promotion of the growth of tomato plants. The widespread ACC deaminase activity in Burkholderia species and the common association of these species with plants suggest that this genus could be a major contributor to plant growth under natural conditions.

We are interested in identifying proteins that interact with the MADS domain protein NMH7 of Medicago sativa. We use an affinity column with a synthetic peptide derived from the MADS domain of NMH7 which has been reported to mediate protein-protein interaction with non-MADS domain interacting proteins. We identified approximately 40 and approximately 80kDa specifically bound proteins as the monomeric and dimeric forms of Fructose-1,6-bisphosphate aldolase cytosolic class I. NiNTA pull down assays revealed that K- and C-terminus regions of NMH7 are not required for the interaction with aldolase. Aldolase enzymatic activity is not required for the interaction with NMH7. NMH7 and aldolase were coimmunoprecipitated from non-inoculated seed and seedlings extracts. Colocalization studies using confocal microscopy showed that aldolase and NMH7 are localized in the cytoplasm and the nucleus of the cortical cells. These data together show that M. sativa aldolase is a novel MADS domain binding protein, and suggest a broader functional repertory for this enzyme, as has been proposed for other glycolytic enzymes.

Salmonella enterica serovar Typhimurium mutants with mutations in the ompS1 and ompS2 genes, which code for quiescent porins, were nevertheless highly attenuated for virulence in a mouse model, indicating a role in pathogenesis. Similarly, a strain with a mutation in the gene coding for LeuO, a positive regulator of ompS2, was also attenuated.

The widespread replicons of repABC and repC families from alpha-proteobacteria share high similarity in their replication initiator proteins (RepC). Here we describe the minimal region required for stable replication of a member of the repC family, the low copy-number plasmid pRmeGR4a from Sinorizobium meliloti GR4. This region contains only two genes: one encoding the initiator protein RepC (46.8 kDa) and other, an antisense RNA (67 nt). Mapping of transcriptional start sites and promoter regions of both genes showed that the antisense RNA is nested within the repC mRNA leader. The constitutively expressed countertranscribed RNA (ctRNA) forms a single stem-loop structure that acts as an intrinsic rho-independent terminator. The ctRNA is a strong trans-incompatibility factor and negative regulator of repC expression. Based on structural and functional similarities between members of the repC and repABC families we propose a model of their evolutionary relationship.

Partial nucleotide sequences of the citrate synthase (gltA) gene from different rhizobia genera were determined. Tree topologies based on this housekeeping gene were similar to that obtained using 16S rRNA sequences. However gltA appeared to be more reliable at determining phylogenetic relationships of closely related taxa. We propose gltA sequences as an additional tool to be used in molecular phylogenetic studies.

Sinorhizobium sp. strain BR816 possesses two nodPQ copies, providing activated sulfate (3'-phosphoadenosine-5'-phosphosulfate [PAPS]) needed for the biosynthesis of sulfated Nod factors. It was previously shown that the Nod factors synthesized by a nodPQ double mutant are not structurally different from those of the wild-type strain. In this study, we describe the characterization of a third sulfate activation locus. Two open reading frames were fully characterized and displayed the highest similarity with the Sinorhizobium meliloti housekeeping ATP sulfurylase subunits, encoded by the cysDN genes. The growth characteristics as well as the levels of Nod factor sulfation of a cysD mutant (FAJ1600) and a nodP1 nodQ2 cysD triple mutant (FAJ1604) were determined. FAJ1600 shows a prolonged lag phase only with inorganic sulfate as the sole sulfur source, compared to the wild-type parent. On the other hand, FAJ1604 requires cysteine for growth and produces sulfate-free Nod factors. Apigenin-induced nod gene expression for Nod factor synthesis does not influence the growth characteristics of any of the strains studied in the presence of different sulfur sources. In this way, it could be demonstrated that the "household" CysDN sulfate activation complex of Sinorhizobium sp. strain BR816 can additionally ensure Nod factor sulfation, whereas the symbiotic PAPS pool, generated by the nodPQ sulfate activation loci, can be engaged for sulfation of amino acids. Finally, our results show that rhizobial growth defects are likely the reason for a decreased nitrogen fixation capacity of bean plants inoculated with cysD mutant strains, which can be restored by adding methionine to the plant nutrient solution.

In the course of a study conducted to isolate genes upregulated by plant cell wall sugars, we identified an arabinose-inducible locus from a transcriptional fusion library of Rhizobium leguminosarum VF39, carrying random insertions of the lacZ transposon Tn5B22. Sequence analysis of the locus disrupted by the transposon revealed a high similarity to uncharacterized malate synthase G genes from Sinorhizobium meliloti, Agrobacterium tumefaciens, and Mesorhizobium loti. This enzyme catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate and CoA and is thought to be a component of the glyoxylate cycle, which allows microorganisms to grow on two carbon compounds. Enzyme assays showed that a functional malate synthase is encoded in the glcB gene of R. leguminosarum and that its expression is induced by arabinose, glycolate, and glyoxylate. An Escherichia coli aceB glcB mutant, complemented with the R. leguminosarum PCR-amplified gene, recovered malate synthase activity. A very similar genome organization of the loci containing malate synthase and flanking genes was observed in R. leguminosarum, S. meliloti, and A. tumefaciens. Pea plants inoculated with the glcB mutant or the wild-type strain showed no significant differences in nitrogen fixation. This is the first report regarding the characterization of a mutant in one of the glyoxylate cycle enzymes in the rhizobia.

Ensifer adhaerens is a soil bacterium that attaches to other bacteria and may cause lysis of these other bacteria. Based on the sequence of its small-subunit rRNA gene, E. adhaerens is related to Sinorhizobium spp. E. adhaerens ATCC 33499 did not nodulate Phaseolus vulgaris (bean) or Leucaena leucocephala, but with symbiotic plasmids from Rhizobium tropici CFN299 it formed nitrogen-fixing nodules on both hosts. The nodule isolates were identified as E. adhaerens isolates by growth on selective media.

A defined insertion mutant of a gene encoding a homolog of the rhizobial C4-dicarboxylate permease (dctA) was constructed in Rhizobium tropici strain CIAT899. This mutant (GA1) was unable to grow on fumarate or malate; however, in contrast with other rhizobial dctA mutants, it retained a limited ability to grow on succinate with ammonia as a nitrogen source. Our results suggest the presence of a novel succinate-specific transport system in R. tropici. Biochemical characterization indicated that this alternative transport system in GAI is active and dependent on an energized membrane. It was also induced by succinate and aspartate, and was repressed by glucose and glycerol. Bean plants inoculated with GA1 showed a reduced nitrogen-fixing ability, achieving only 29% of the acetylene reduction activity determined in CIAT899 strain nodules, 33 days after inoculation. Also, bean plants inoculated with GA1 had reduced shoot dry weight compared with plants inoculated with the wild-type strain.

The genetic structure of Bradyrhizobium isolates recovered from three Lupinus species (Lupinus campestris, Lupinus montanus, and Lupinus exaltatus) grown in Mexico was examined. Among 41 Bradyrhizobium isolates, 18 electrophoretic types (ETs) were distinguished by multilocus enzyme electrophoresis of five metabolic enzymes. The mean genetic diversity, 0.64, indicated that there was great genetic diversity in the population sampled. Most isolates (63%) fell into two closely related clusters (clusters I and II) and were the types most frequently isolated from the root nodules of L. montanus and L. campestris. ET cluster III isolates were frequent nodule occupants of L. exaltatus. The isolates also were assigned to three main groups by using Curie point pyrolysis mass spectrometry. In general, the multilocus enzyme electrophoretic data and pyrolysis mass spectrometric data agreed. We determined the 16S rRNA sequences of representative Lupinus isolates and of Bradyrhizobium japonicum USDA 6T and found that the lupine isolates were highly related to the B. japonicum type strain, although not all B. japonicum type strains (subcultures maintained in different bacterial collections) had identical small-subunit rRNA.

We determined the activities of selected enzymes involved in carbon metabolism in free-living cells of Rhizobium tropici CFN299 grown in minimal medium with different carbon sources and in bacteroids of the same strain. The set of enzymatic activities in sucrose-grown cells suggests that the pentose phosphate pathway, with the participation of the Entner-Doudoroff pathway, is probably the primary route for sugar catabolism. In glutamate- and malate-grown cells, high activities of the gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-6-phosphate aldolase, and fructose bisphosphatase) were detected. In bacteroids, isolated in Percoll gradients, the levels of activity for many of the enzymes measured were similar to those of malate-grown cells, except that higher activities of glucokinase, glucose-6-phosphate dehydrogenase, and NAD-dependent phosphogluconate dehydrogenase were detected. Phosphoglucomutase and UDP glucose pyrophosphorylase showed high and constant levels under all growth conditions and in bacteroids.