The composite genome of the legume symbiont Sinorhizobium meliloti.

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

Codon Usage Heterogeneity in the Multipartite Prokaryote Genome: Selection-Based Coding Bias Associated with Gene Location, Expression Level, and Ancestry.

Prokaryotes represent an ancestral lineage in the tree of life and constitute optimal resources for investigating the evolution of genomes in unicellular organisms. Many bacterial species possess multipartite genomes offering opportunities to study functional variations among replicons, how and where new genes integrate into a genome, and how genetic information within a lineage becomes encoded and evolves. To analyze these issues, we focused on the model soil bacterium Sinorhizobium meliloti, which harbors a chromosome, a chromid (pSymB), a megaplasmid (pSymA), and, in many strains, one or more accessory plasmids. The analysis of several genomes, together with 1.4 Mb of accessory plasmid DNA that we purified and sequenced, revealed clearly different functional profiles associated with each genomic entity. pSymA, in particular, exhibited remarkable interstrain variation and a high density of singletons (unique, exclusive genes) featuring functionalities and modal codon usages that were very similar to those of the plasmidome. All this evidence reinforces the idea of a close relationship between pSymA and the plasmidome. Correspondence analyses revealed that adaptation of codon usages to the translational machinery increased from plasmidome to pSymA to pSymB to chromosome, corresponding as such to the ancestry of each replicon in the lineage. We demonstrated that chromosomal core genes gradually adapted to the translational machinery, reminiscent of observations in several bacterial taxa for genes with high expression levels. Such findings indicate a previously undiscovered codon usage adaptation associated with the chromosomal core information that likely operates to improve bacterial fitness. We present a comprehensive model illustrating the central findings described here, discussed in the context of the changes occurring during the evolution of a multipartite prokaryote genome.IMPORTANCE Bacterial genomes usually include many thousands of genes which are expressed with diverse spatial-temporal patterns and intensities. A well-known evidence is that highly expressed genes, such as the ribosomal and other translation-related proteins (RTRPs), have accommodated their codon usage to optimize translation efficiency and accuracy. Using a bioinformatic approach, we identify core-genes sets with different ancestries, and demonstrate that selection processes that optimize codon usage are not restricted to RTRPs but extended at a genome-wide scale. Such findings highlight, for the first time, a previously undiscovered adaptation strategy associated with the chromosomal-core information. Contrasted with the translationally more adapted genes, singletons (i.e., exclusive genes, including those of the plasmidome) appear as the gene pool with the less-ameliorated codon usage in the lineage. A comprehensive summary describing the inter- and intra-replicon heterogeneity of codon usages in a complex prokaryote genome is presented.

Identification and characterization of a nodH ortholog from the alfalfa-nodulating Or191-like rhizobia.

Nodulation of Medicago sativa (alfalfa) is known to be restricted to Sinorhizobium meliloti and a few other rhizobia that include the poorly characterized isolates related to Rhizobium sp. strain Or191. Distinctive features of the symbiosis between alfalfa and S. meliloti are the marked specificity from the plant to the bacteria and the strict requirement for the presence of sulfated lipochitooligosaccharides (Nod factors [NFs]) at its reducing end. Here, we present evidence of the presence of a functional nodH-encoded NF sulfotransferase in the Or191-like rhizobia. The nodH gene, present in single copy, maps to a high molecular weight megaplasmid. As in S. meliloti, a nodF homolog was identified immediately upstream of nodH that was transcribed in the opposite direction (local synteny). This novel nodH ortholog was cloned and shown to restore both NF sulfation and the Nif+Fix+ phenotypes when introduced into an S. meliloti nodH mutant. Unexpectedly, however, nodH disruption in the Or191-like bacteria did not abolish their ability to nodulate alfalfa, resulting instead in a severely delayed nodulation. In agreement with evidence from other authors, the nodH sequence analysis strongly supports the idea that the Or191-like rhizobia most likely represent a genetic mosaic resulting from the horizontal transfer of symbiotic genes from a sinorhizobial megaplasmid to a not yet clearly identified ancestor.

Cooperative Action of Rhizobium meliloti Nodulation and Infection Mutants during the Process of Forming Mixed Infected Alfalfa Nodules.

Alfalfa plants co-inoculated with Rhizobium meliloti nodulation (Nod-) and infection mutants deficient in exopolysaccharide production (Inf-EPS-) formed mixed infected nodules that were capable of fixing atmospheric nitrogen. The formation of infected nodules was dependent on close contact between the inoculation partners. When the partners were separated by a filter, empty Fix- nodules were formed, suggesting that infection thread formation in alfalfa is dependent on signals from the nodulation and infection genes. In mixed infected nodules, both nodulation and infection mutants colonized the plant cells and differentiated into bacteroids. The formation of bacteroids was not dependent on cell-to-cell contact between the mutants. Immunogold/silver staining revealed that the ratio of the two mutants varied considerably in colonized plant cells following mixed inoculation. The introduction of an additional nif/fix mutation into one of the inoculation partners did not abolish nitrogen fixation in mixed infected nodules. The expression of nif D::lacZ fusions additionally demonstrated that mutations in the nodulation and infection genes did not prevent the nif genes from being expressed in the mutant bacteroids.

[Will carotid thromboendarterectomy remain competitive? Influence of intraoperative duplex ultrasound quality control]. / Wird die Karotischirurgie konkurrenzfähig bleiben? Einfluss intraoperativer duplexsonographischer Qualitätskontrolle.

BACKGROUND: Thromboendarterectomy (TEA) and stenting are in competition for treatment of carotid artery lesions. Both treatment modalities have to improve significantly. The goal of the study was to evaluate the influence of routine intraoperative duplex ultrasound examination. METHODS: In a continuous prospective study, 575 patients underwent 620 carotid operations. Intraoperative duplex ultrasound examination was performed prior to wound closure: 9.5% had significant contralateral ICA stenoses and 6.7% ICA occlusion; 8.5% presented special lesions. An eversion TEA was performed in 20.5% while 78.5% underwent conventional TEA with patch plasty and graft interposition in 1%. Intraoperative quality control revealed unexpected lesions in 10% requiring immediate repair. RESULTS: The combined morbidity/mortality rate (MMR) of the total series was 2.6%. Women had an elevated risk (4.2%) in comparison to men (1.9%). The risk of elder patients (>75 years, n=151) was remarkably low. The neurological complication rate of the total series was 1.6% and the incidence of major strokes 1.1%. CONCLUSIONS: Routine intraoperative duplex ultrasound examination of the carotid reconstruction allows early diagnosis and immediate correction of morphologic as well as hemodynamic lesions. Competing with stent placement a further reduction of complications of carotid TEA seems to be possible and necessary.

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa.

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101-pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1beta) and RK2 (IncP-1alpha), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.

A consolidated analysis of the physiologic and molecular responses induced under acid stress in the legume-symbiont model-soil bacterium Sinorhizobium meliloti.

Abiotic stresses in general and extracellular acidity in particular disturb and limit nitrogen-fixing symbioses between rhizobia and their host legumes. Except for valuable molecular-biological studies on different rhizobia, no consolidated models have been formulated to describe the central physiologic changes that occur in acid-stressed bacteria. We present here an integrated analysis entailing the main cultural, metabolic, and molecular responses of the model bacterium Sinorhizobium meliloti growing under controlled acid stress in a chemostat. A stepwise extracellular acidification of the culture medium had indicated that S. meliloti stopped growing at ca. pH 6.0-6.1. Under such stress the rhizobia increased the O2 consumption per cell by more than 5-fold. This phenotype, together with an increase in the transcripts for several membrane cytochromes, entails a higher aerobic-respiration rate in the acid-stressed rhizobia. Multivariate analysis of global metabolome data served to unequivocally correlate specific-metabolite profiles with the extracellular pH, showing that at low pH the pentose-phosphate pathway exhibited increases in several transcripts, enzymes, and metabolites. Further analyses should be focused on the time course of the observed changes, its associated intracellular signaling, and on the comparison with the changes that operate during the sub lethal acid-adaptive response (ATR) in rhizobia.

Denaturation map of the circular mitochondrial genome of Neurospora crassa.

A denaturation map of mitochondrial DNA from the wild type strain 5256 of Neurospora crassa was constructed by computer analysis of the contour length distribution of single- and double-stranded regions of nineteen circular and three full length linear molecules after partial denaturation. The data suggest that mitochondrial DNA in this strain is a homogeneous population of a circular molecule of molecular weight 41 - 10(6) with an asymmetric distribution of AT-rich regions, and that linear molecules derive from this genome by random breaks during isolation.

Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae.

The complete nucleotide sequence (24,206 base-pairs) of the Klebsiella pneumoniae gene region for nitrogen fixation (nif) is presented. Coding regions corresponding to the 19 known nif genes (including nifW and nifZ) could be identified. An additional open reading frame of 216 base-pairs, called nifT, was detected between nifK and nifY. Search for transcriptional signal structures revealed some unusual features: (1) several possible NifA-binding motifs are present in the intergenic regions between nifJ and nifH as well as between nifX and nifU; (2) a perfect NifA-binding motif, preceding the nifENX promoter, is located within an inverted repeat structure; (3) structures resembling the consensus nif promoter are found within the coding regions of nifW and nifZ and, together with a NifA-binding motif, in nifN. Typical rho-independent termination structures were detected only downstream from the nifHDKTY and the nifBQ operons. Analysis of the deduced amino acid sequences revealed the presence of two Cys-X2-Cys-X2-Cys-X3-Cys-Pro clusters in the pyruvate-flavodoxin oxidoreductase NifJ. This arrangement of cysteine residues is normally present only in ferredoxins. A high degree of homology between the two gene products (NifE and NifN) involved in iron-molybdenum cofactor biosynthesis and the two nitrogenase component I structural proteins (NifD and NifK) was found. All four proteins are characterized by the conserved motif His-Gly-X2-Gly-Cys, which may play a role in binding the iron-molybdenum cofactor.

The Metabolites of the Herbicide L-Phosphinothricin (Glufosinate) (Identification, Stability, and Mobility in Transgenic, Herbicide-Resistant, and Untransformed Plants).

The metabolism of the herbicide L-phosphinothricin (L-Pt) was analyzed in tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), and carrot (Daucus carota). In transgenic, Pt-resistant plants expressing the Pt-N-acetyltransferase gene (pat), L-Pt was acetylated, resulting in two forms of N-acetyl-Pt (ac-Pt). In transgenic plants expressing only low pat-encoded acetylating activity as well as in genetically unmodified plants, three metabolic compounds 4-methylphosphinico-2-oxo-butanoic acid, 3-methylphosphinico-propanoic acid (MPP), and 4-methylphosphinico-2-hydroxy-butanoic acid (MHB) were identified. Hence, the transgene-encoded acetylation of L-Pt competes with a plant-specific degradation. The compounds MPP, MHB, and ac-Pt were found to be the final, stable products of the plant's metabolic pathways. The mobility of these stable compounds in the plant was investigated: L-Pt as well as the derived metabolites were found to be preferentially transported to the upper regions of the plant.

Successful heterologous expression of a novel chitinase identified by sequence analyses of the metagenome from a chitin-enriched soil sample.

Chitin and its derivative chitosan are abundant natural polysaccharides with many potential industrial applications. Metagenomic analysis of chitin-enriched soil samples using the Roche Genome Sequencer FLX platform led to the identification of several novel genes for chitin and chitosan modifying enzymes (CCMEs) which may be used to produce novel chitosans. The sequencing approach yielded 2,281,090 reads with an average length of 378 bp amounting to a total sequence information of approximately 851 Mb. Assembly of the obtained sequences comprised 699,710 reads representing 30.68% of all reads. A total of 6625 contigs larger than 500 bp containing 16,289 predicted genes are included in the assembly. Taxonomic profiling of the indigenous microbial community by applying the software CARMA revealed that 96.1% of the reads were of bacterial origin including 17% assigned to the family Xanthomonadaceae. Several putative genes encoding CCMEs were identified by comparison against the GenBank database, inclusive a full-length chitinase gene which was codon optimized for Escherichia coli and heterologously synthesized as a Strep-tagged protein in E. coli Rosetta 2 using the pET vector system. Approximately 5mg of the novel active chitinase was purified as demonstrated by dot assay analysis using glycol chitin as a substrate. Next generation metagenomic sequencing, thus, emerges as a new and powerful tool for the identification of potentially novel biocatalysts of biotechnological value.

The sucrose synthase gene is predominantly expressed in the root nodule tissue of Vicia faba.

To analyze nodule-specific gene expression in broadbean, we have isolated and sequenced sucrose synthase (SUCS) cDNAs from a broadbean nodule-specific cDNA library. The most 5' sequences identified from these partial cDNAs were used as a molecular probe to isolate a full-length sucrose synthase transcript sequence from a cDNA library derived from broadbean nodule mRNA. This cDNA (VfSUCS) contained a reading frame of 2,418 bp, coding for a protein of 806 amino acids with a deduced molecular weight of 92.5 kDa. The DNA as well as the deduced amino acid sequence displayed substantial homologies (68-95%) to other plant SUCS sequences. Northern and RNA dot blot experiments demonstrated that this gene is strongly expressed in the broadbean nodule tissue. An at least 10-fold lower VfSUCS expression could be detected in the uninfected root, hypocotyl, stem, and flower tissues of broadbean, whereas only traces of VfSUCS transcripts were recognizable in the broadbean leaf tissues. VfSUCS transcripts could not be detected in mature seeds of broadbean. Because of this significantly nodule-amplified type of expression, we refer to VfSUCS as a nodulin gene and propose to designate it VfNOD93 (Nuf-93) for the sucrose synthase enzyme).

The Vicia faba lipoxygenase gene VfLOX1 is expressed in the root nodule parenchyma.

A full-length cDNA encoding the broad bean lipoxygenase VfLOX1 was isolated from a nodule cDNA library. The VfLOX1 gene was strongly expressed in nodules, and only weakly in roots. VfLOX1 transcripts were localized in the nodule parenchyma and in the cells surrounding the root stele.

The Vicia faba leghemoglobin gene VfLb29 is induced in root nodules and in roots colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum.

To investigate similarities between symbiotic interactions of broad bean (Vicia faba) with rhizobia and mycorrhizal fungi, plant gene expression induced by both microsymbionts was compared. We demonstrated the exclusive expression of 19 broad bean genes, including VfENOD2, VfENOD5, VfENOD12 and three different leghemoglobin genes, in root nodules. In contrast, the leghemoglobin gene VfLb29 was found to be induced not only in root nodules, but also in broad bean roots colonized by the mycorrhizal fungus Glomus fasciculatum. In uninfected roots, none of the 20 nodulin transcripts investigated was detectable. VfLb29 has an unusually low sequence homology with all other broad bean leghemoglobins as well as with leghemoglobins from other legumes. It can be regarded as a novel kind of leghemoglobin gene not described until now and the induction of which is common to symbiotic interactions of broad bean with both Rhizobium and a mycorrhizal fungus.

Genetic analysis of the Rhizobium meliloti exoYFQ operon: ExoY is homologous to sugar transferases and ExoQ represents a transmembrane protein.

The nucleotide sequence of a 4.8-kb ClaI-EcoRI DNA fragment of megaplasmid 2 of Rhizobium meliloti Rm2011 involved in succinoglucan (EPS I) synthesis and nodule infection was determined. Four open reading frames (ORFs) were identified on this fragment. A mutational analysis revealed that these ORFs represent genes that were termed exoX, exoY, exoF, and exoQ. The locations of transposon insertions in these exo genes were determined at the nucleotide level. Plasmid integration mutagenesis revealed that the genes exoY, exoF, and exoQ are organized in an operon. The exoX gene running in opposite direction forms a monocistronic transcriptional unit. The exoX gene was shown to negatively influence the amount of EPS I synthesized. The exoY gene is coding for a membrane associated protein homologous to the C-terminal part of the Xanthomonas campestris glucosyltransferase GumD and the Salmonella typhimurium galactose transferase RfbP. ExoF, a probable periplasmatic protein, is nearly identical to the protein encoded by ORF1 of Rhizobium sp. strain NGR234. ExoQ is most probably a membrane associated protein as deduced by its hydrophobic structural features. All three genes of the exoYFQ operon were shown to be essential for succinoglucan synthesis and nodule infection.

Analysis of the Rhizobium meliloti genes exoU, exoV, exoW, exoT, and exoI involved in exopolysaccharide biosynthesis and nodule invasion: exoU and exoW probably encode glucosyltransferases.

Sequence analysis of a 5.780-kb DNA fragment originating from megaplasmid 2 of Rhizobium meliloti 2011 involved in biosynthesis of exopolysaccharide I (EPS I) and invasion of alfalfa nodules revealed the presence of five exo genes designated exoU, exoV, exoW, exoT, and exoI. ExoT resembled transmembrane proteins, whereas ExoI displayed a characteristic signal peptide. Sequence comparisons with several polysaccharide-polymerizing enzymes of both prokaryotic and eukaryotic origin indicated that exoW and exoU encode glucosyltransferases. Moreover, ExoV displayed weak homologies to the ExoO, ExoA, ExoL, and ExoM proteins of R. meliloti, which are also discussed as glucosyltransferases. Using exo-lacZ transcription fusions in connection with plasmid integration mutagenesis, promoters were identified in front of exoI, exoT, exoW, exoV, and exoU.R. meliloti 2011 strains with mutations in exoT, exoW, exoV, and exoU produced no detectable EPS I and were unable to infect alfalfa nodules, whereas exoI mutants synthesized a reduced amount of EPS I and did infect alfalfa nodules.

Molecular analysis of the Rhizobium meliloti mucR gene regulating the biosynthesis of the exopolysaccharides succinoglycan and galactoglucan.

The Rhizobium meliloti Tn5 mutant Rm3131, producing galactoglucan (EPS II) instead of succinoglycan (EPS I), was complemented by a 3.6-kb EcoRI-fragment of the Rhizobium meliloti genome. Sequencing of this fragment revealed six open reading frames (ORFs). The ORF found to be affected in the mutant Rm3131 codes for a putative protein of 15.7 kDa and forms a monocistronic transcriptional unit. Further genetic analysis revealed that the gene mutated in Rm3131 is identical to the previously described R. meliloti mucR gene (H. Zhan, S.B. Levery, C. C. Lee, and J.A. Leigh, 1989, Proc. Natl. Acad. Sci. USA 86:3055-3059). By hybridization it was shown that a mucR homologous gene is present in several rhizobacteria. The deduced amino acid sequence of MucR showed nearly 80% identity to the Agrobacterium tumefaciens Ros protein, a negative regulator of vir genes and necessary for succinoglycan production. MucR contains like Ros a putative zinc finger sequence of the C2H2 type. Transcriptional fusions of genes for EPS I and EPS II synthesis, the so-called exo and exp genes, with the marker gene lacZ were used to delineate the role of mucR for exo and exp gene expression. It was found that exp genes are negatively regulated by MucR on the transcriptional level, whereas a posttranscriptional regulation by MucR is assumed for exo genes. Furthermore, mucR is negatively regulating its own transcription.

A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing.

A set of plasmid vectors was developed which allows fast sequencing by the chemical degradation method. These high-copy-number vectors are derivatives of the plasmid pUC8 containing different multiple-purpose cloning sites flanked by unique recognition sequences for the restriction enzymes BstEII, Tth111I and Eco81I as sites for end-labelling DNA. Due to their partially asymmetric recognition sequences, each of these three restriction sites can be singly end-labelled by a filling-in reaction with selected nucleotides. This allows easy single end-labelling of any cloned DNA fragment for sequencing by the chemical degradation method without any isolation and purification step after the labelling reaction. In addition, the nucleotide sequence of the complementary strand from the same end can be determined by the dideoxy chain termination procedure using the universal M13 primers. In most of the new vectors, the reading frame of the lacZ' gene is retained, allowing identification of cloned fragments.

Cloning of a phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Streptomyces lividans and Escherichia coli.

Phosphinothricin-tripeptide (Ptt), also known as bialaphos, contains phosphinothricin (Pt), a potent inhibitor of glutamine synthetase. A 4.0-kb Bam HI fragment coding for Ptt resistance was cloned in Streptomyces lividans TK23. The fragment was isolated from a Ptt-resistant mutant of Streptomyces viridochromogenes Tü494. Subcloning experiments revealed that Ptt resistance can be assigned to a 0.8-kb Bg/II fragment. This fragment was shown to include the Ptt-resistance promoter. Subcloning this fragment downstream from the lacZ promoter conferred Ptt resistance to Escherichia coli JM83 in one of the two possible orientations. Biochemical investigations revealed that the Bg/II fragment codes for a Pt N-acetyltransferase.

A new family of RSF1010-derived expression and lac-fusion broad-host-range vectors for gram-negative bacteria.

A series of broad-host-range expression and lac fusion vectors, based on RSF1010 derivatives, was constructed. The expression vectors contain various promoters (pNm, plac, ptac and pS1) for expression of foreign genes. The efficiency of the promoters was determined in Escherichia coli, Rhizobium meliloti, Rhizobium leguminosarum and Pseudomonas putida by beta-galactosidase activity measurements. Of the promoters assayed in E. coli, the most effective is the tac promoter, whereas in soil bacteria the appropriate promoter for overexpression of foreign genes is the NmR promoter. The GmR gene, serving as a selectable marker for the plasmids, was efficiently expressed in R. meliloti as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thus, pGm was also used to construct an expression vector. The translational fusion vectors allow the identification and characterization of promoter-carrying cloned fragments on the translational level, whereas the transcriptional fusion vectors can be used to identify and to study promoters on cloned fragments. All lac fusion vectors contain the E. coli lacZ gene or the complete lac operon facilitating quantification of expression.