Molecular and genetic characterization of the rhizopine catabolism (mocABRC) genes of Rhizobium meliloti L5-30

Rhizopine (l-3-O-methyl-scyllo-inosamine, 3-O-MSI) is a symbiosis-specific compound, which is synthesized in nitrogen-fixing nodules of Medicago sativa induced by Rhizobium meliloti strain L5–30. 3-O-MSI is thought to function as an unusual growth substrate for R. meliloti L5–30, which carries a locus (mos) responsible for its synthesis closely linked to a locus (moc) responsible for its degradation. Here, the essential moc genes were delimited by Tn5 mutagenesis and shown to be organized into two regions, separated by 3 kb of DNA. The DNA sequence of a 9-kb fragment spanning the two moc regions was determined, and four genes were identified that play an essential role in rhizopine catabolism (mocABC and mocR). The analysis of the DNA sequence and the amino acid sequence of the deduced protein products revealed that MocA resembles NADH-dependent dehydrogenases. MocB exhibits characteristic features of periplasmic-binding proteins that are components of high-affinity transport systems. MocC does not share significant homology with any protein in the database. MocR shows homology with the GntR class of bacterial regulator proteins. These results suggest that the mocABC genes are involved in the uptake and subsequent degradation of rhizopine, whereas mocR is likely to play a regulatory role.     

Characterization of genes for synthesis and catabolism of a new rhizopine induced in nodules by Rhizobium meliloti Rm220-3: extension of the rhizopine concept.

Rhizopines are selective growth substrates synthesized in nodules only by strains of rhizobia capable of their catabolism. We report the isolation and study of genes for the synthesis and catabolism of a new rhizopine, scyllo-inosamine (sIa), from alfalfa nodules induced by Rhizobium meliloti Rm220-3. This compound is similar in structure to the previously described rhizopine 3-O-methyl-scyllo-inosamine from R. meliloti L5-30 (P.J. Murphy, N. Heycke, Z. Banfalvi, M.E. Tate, F.J. de Bruijn, A. Kondorosi, J. Tempé, and J. Schell, Proc. Natl. Acad. Sci. USA 84:493-497, 1987). The synthesis (mos) and catabolism (moc) genes for the Rm220-3 rhizopine are closely linked and located on the nod-nif Sym plasmid. The mos genes are directly controlled by the NifA/NtrA regulatory system. A comparison of the sequence of the 5' regions of the two mos loci shows very extensive conservation of sequence as well as strong homology to the nifH coding region. Restriction mapping and hybridization to DNA from the four open reading frames (ORFs) of the L5-30 mos locus indicate the absence of mosA and presence of the other three ORFs (ORF1 and mosB and -C) in Rm220-3. We suggest that the L5-30 mosA gene product is involved in the conversion of scyllo-inosamine to 3-O-methyl-scyllo-inosamine. Restriction fragment length polymorphism analysis of the moc regions of both strains shows that they are very similar. Regulation studies indicate that the moc region is not controlled by the common regulatory gene nifA, ntrA, and ntrC. We discuss the striking similarities in gene structure, location, and regulation between these two rhizopine loci in relation to the rhizopine concept.     

A 'bacteriocin PCR array' for identification of bacteriocin-related structural genes in lactic acid bacteria

Bacteriocins have been identified in many strains of lactic acid bacteria (LAB) which are a source of natural food preservatives and microbial inhibitors. Our objectives were to use a PCR array of primers to identify bacteriocin structural genes in Bac⁺ LAB. DNA sequence homology at the 5′- and 3′-ends of the various structural genes indicated that non-specific priming may allow PCR amplification of heterologous bacteriocin genes. Successful amplification was obtained by real-time PCR and confirmed by melting curve and agarose gel analysis. Sequence information specific to targeted bacteriocin structural genes from the intra-primer regions of amplimers was compared to sequences residing in GenBank. The bacteriocin PCR array allowed the successful amplification of bacteriocin structural genes from strains of Lactobacillus, Lactococcus, and Pediococcus including one whose amino acid sequence was unable to be determined by Edman degradation analysis. DNA sequence analysis identified as many as 3 bacteriocin structural genes within a given strain, identifying ten unique bacteriocin sequences that were previously uncharacterized (partial homology) and one that was 100% identical to sequences in GenBank. This study provides a rapid approach to sequence and identify bacteriocin structural genes among Bac⁺ LAB using a microplate bacteriocin PCR array.     

Rhizobium (Sinorhizobium) meliloti phn Genes: Characterization and Identification of Their Protein Products

In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA from Rhizobium (Sinorhizobium) meliloti that contains homologues of the E. coli phnG, -H, -I, -J, and -K genes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the R. meliloti and E. coli phn genes and their predicted products gave new information about C-P lyase. The putative R. meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by R. meliloti grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.     

Nucleotide Sequence Homology Exists between the Chloroplast and Nuclear Ribosomal DNAs of Euglena gracilis

The nuclear and chloroplast ribosomal DNAs from Euglena were shown to have specific regions of nucleotide sequence homology. The regions of homology were identified by hybridization of restriction endonuclease DNA fragments of cloned chloroplast and nuclear ribosomal DNAs to one another. The regions of homology between these two ribosomal DNAs were in that part of the genes that code for the 3′ end of the small rRNAs (16S and 19S) and near or at the DNA sequences coding for the 5S RNAs. The nucleotide sequence homology between these regions was estimated to be approximately 94% by the melting point depression of a hybrid formed between the two ribosomal DNAs.     

<Emphasis Type="Italic">tolC</Emphasis> mutant of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> strain CXM1-188 fails to establish effective symbiosis with alfalfa

The TolC mutant Tr63 of Sinorhizobium meliloti was generated by random Tn5 mutagenesis in the effective strain CXM1-188. The mutant did not produce fluorescent halos in UV light on the LB medium containing Calcofluor white, which suggests that modification occurred in the production of exopolysaccharide EPS1. Mutant Tr63 also manifested nonmucoidness both on minimal and low-phosphate MOPS media, and this was most likely connected with the absence of the second exopolysaccharide of S. meliloti (EPS2). The mutant was defective in symbiosis with alfalfa and formed on roots of host plants Medicago sativa and M. truncatula white round Fix^? nodules or nodules of irregular shape. These nodules possessed the structure usually described for nodules of EPS1 mutants. According to the data of sequencing a DNA fragment of the mutant adjacent to the transposon, Tr63 contained a Tn5 insertion in gene SMc02082 located on the S. meliloti chromosome. This gene encodes the protein sharing homology with the TolC protein, a component of a type I secretion system responsible for the export of protein toxins and proteases in Gram-negative bacteria. The presence of proteins ExsH (endoglycanase of EPS1) and protein ExpE1 (essential for excretion of EPS2), which are known to be exported by the type I secretion system, was tested in cultural supernatants of mutant Tr63 and the parental strain by polyclonal antiserum analysis. It was ascertained that secretory proteins ExsH and ExpE1 are absent in the culture medium of mutant Tr63. The TolC protein of S. meliloti is assumed to be involved in the excretion of proteins ExsH and ExpE1.     

The 172-kb genomic DNA region of the O. rufipogon yld1.1 locus: comparative sequence analysis with O. sativa ssp. japonica and O. sativa ssp. indica

Common wild rice (Oryza rufipogon) plays an important role by contributing to modern rice breeding. In this paper, we report the sequence and analysis of a 172-kb genomic DNA region of wild rice around the RM5 locus, which is associated with the yield QTL yld1.1. Comparative sequence analysis between orthologous RM5 regions from Oryza sativa ssp. japonica, O. sativa ssp. indica and O. rufipogon revealed a high level of conserved synteny in the content, homology, structure, orientation, and physical distance of all 14 predicted genes. Twelve of the putative genes were supported by matches to proteins with known function, whereas two were predicted by homology to rice and other plant expressed sequence tags or complementary DNAs. The remarkably high level of conservation found in coding, intronic and intergenic regions may indicate high evolutionary selection on the RM5 region. Although our analysis has not defined which gene(s) determine the yld1.1 phenotype, allelic variation and the insertion of transposable elements, among other nucleotide changes, represent potential variation responsible for the yield QTL. However, as suggested previously, two putative receptor-like protein kinase genes remain the key suspects for yld1.1. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of Mice with Single and Multiple Copies of Transgenes Reveals a Novel Arrangement for the λ5-VpreB1 Locus Control Region

The murine λ5-V_preB1 locus encodes two proteins that form part of the pre-B-cell receptor and play a key role in B-lymphocyte development. We have identified a locus control region (LCR) which is responsible for coordinate activation of both genes in pre-B cells. Analysis of mice with single and multiple copies of transgenes shows a clear difference in the expression behavior of the genes depending on the transgene copy number. While expression of both λ5 and V_preB1 in single- and two-copy integrations requires the presence of a set of DNase I hypersensitive sites located 3′ of the λ5 gene, small fragments containing the genes have LCR activity when arranged in multiple-copy tandem arrays, indicating that additional components of the LCR are located within or close to the genes. The complete LCR is capable of driving efficient copy-dependent expression of a λ5 gene in pre-B cells even when it is integrated into centomeric γ-satellite DNA. The finding that activation of expression of the locus by positively acting factors is fully dominant over the silencing effect of heterochromatin has implications for models for chromatin-mediated gene silencing during B-cell development.     

A New Genetic Locus in Sinorhizobium meliloti Is Involved in Stachydrine Utilization

Stachydrine, a betaine released by germinating alfalfa seeds, functions as an inducer of nodulation genes, a catabolite, and an osmoprotectant in Sinorhizobium meliloti. Two stachydrine-inducible genes were found in S. meliloti 1021 by mutation with a Tn5-luxAB promoter probe. Both mutant strains (S10 and S11) formed effective alfalfa root nodules, but neither grew on stachydrine as the sole carbon and nitrogen source. When grown in the absence or presence of salt stress, S10 and S11 took up [¹⁴C]stachydrine as well as wild-type cells did, but neither used stachydrine effectively as an osmoprotectant. In the absence of salt stress, both S10 and S11 took up less [¹⁴C]proline than wild-type cells did. S10 and S11 appeared to colonize alfalfa roots normally in single-strain tests, but when mixed with the wild-type strain, their rhizosphere counts were reduced more than 50% (P ≤ 0.01) relative to the wild type. These results suggest that stachydrine catabolism contributes to root colonization. DNA sequence analysis identified the mutated locus in S11 as putA, and the luxAB fusion in that gene was induced by proline as well as stachydrine. DNA that restored the capacity of mutant S10 to catabolize stachydrine contained a new open reading frame, stcD. All data are consistent with the concept that stcD codes for an enzyme that produces proline by demethylation of N-methylproline, a degradation product of stachydrine.     

Sequence Analysis of the 144-Kilobase Accessory Plasmid pSmeSM11a, Isolated from a Dominant Sinorhizobium meliloti Strain Identified during a Long-Term Field Release Experiment

The genome of Sinorhizobium meliloti type strain Rm1021 consists of three replicons: the chromosome and two megaplasmids, pSymA and pSymB. Additionally, many indigenous S. meliloti strains possess one or more smaller plasmids, which represent the accessory genome of this species. Here we describe the complete nucleotide sequence of an accessory plasmid, designated pSmeSM11a, that was isolated from a dominant indigenous S. meliloti subpopulation in the context of a long-term field release experiment with genetically modified S. meliloti strains. Sequence analysis of plasmid pSmeSM11a revealed that it is 144,170 bp long and has a mean G+C content of 59.5 mol%. Annotation of the sequence resulted in a total of 160 coding sequences. Functional predictions could be made for 43% of the genes, whereas 57% of the genes encode hypothetical or unknown gene products. Two plasmid replication modules, one belonging to the repABC replicon family and the other belonging to the plasmid type A replicator region family, were identified. Plasmid pSmeSM11a contains a mobilization (mob) module composed of the type IV secretion system-related genes traG and traA and a putative mobC gene. A large continuous region that is about 42 kb long is very similar to a corresponding region located on S. meliloti Rm1021 megaplasmid pSymA. Single-base-pair deletions in the homologous regions are responsible for frameshifts that result in nonparalogous coding sequences. Plasmid pSmeSM11a carries additional copies of the nodulation genes nodP and nodQ that are responsible for Nod factor sulfation. Furthermore, a tauD gene encoding a putative taurine dioxygenase was identified on pSmeSM11a. An acdS gene located on pSmeSM11a is the first example of such a gene in S. meliloti. The deduced acdS gene product is able to deaminate 1-aminocyclopropane-1-carboxylate and is proposed to be involved in reducing the phytohormone ethylene, thus influencing nodulation events. The presence of numerous insertion sequences suggests that these elements mediated acquisition of accessory plasmid modules.     

The Sinorhizobium meliloti Nutrient-Deprivation-Induced Tyrosine Degradation Gene hmgA Is Controlled by a Novel Member of the arsR Family of Regulatory Genes

The regulation of the nutrient-deprivation-induced Sinorhizobium meliloti homogentisate dioxygenase (hmgA) gene, involved in tyrosine degradation, was examined. hmgA expression was found to be independent of the canonical nitrogen regulation (ntr) system. To identify regulators of hmgA, secondary mutagenesis of an S. meliloti strain harboring a hmgA-luxAB reporter gene fusion (N4) was carried out using transposon Tn1721. Two independent Tn1721 insertions were found to be located in a positive regulatory gene (nitR), encoding a protein sharing amino acid sequence similarity with proteins of the ArsR family of regulators. NitR was found to be a regulator of S. meliloti hmgA expression under nitrogen deprivation conditions, suggesting the presence of a ntr-independent nitrogen deprivation regulatory system. nitR insertion mutations were shown not to affect bacterial growth, nodulation of Medicago sativa (alfalfa) plants, or symbiotic nitrogen fixation under the physiological conditions examined. Further analysis of the nitR locus revealed the presence of open reading frames encoding proteins sharing amino acid sequence similarities with an ATP-binding phosphonate transport protein (PhnN), as well as transmembrane efflux proteins.     

New Recombination Methods for Sinorhizobium meliloti Genetics

The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative α-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.     

Metabolism of Tryptophan and Tryptophan Analogs by Rhizobium meliloti

The alfalfa symbiont Rhizobium meliloti Rm1021 produces indole-3-acetic acid in a regulated manner when supplied with exogenous tryptophan. Mutants with altered response to tryptophan analogs still produce indole-3-acetic acid, but are Fix⁻ because bacteria do not fully differentiate into the nitrogen-fixing bacteriod form. These mutations are in apparently essential genes tightly linked to a dominant streptomycin resistance locus.     

Cloning, Characterization, and Possible Origin of the Prevotella loescheii dnaK Homolog

Heat shock proteins play an important role in bacterial survival and response to environmental stress. We cloned the Prevotella loescheii HSP70 homolog (dnaK) and characterized the coding sequence, regulatory regions, and evolutionary relationships to other bacteria. Predicted proteins encoded by the P. loescheii dnaK homolog (open reading frame ORF-1) and two downstream coding regions, ORF-2 and ORF-3, are highly homologous to the proteins encoded by ORF-4 (dnaK), ORF-5, and ORF-6 from the dnaK region of Porphyromonas gingivalis. The dnaK promoter resembles other HSP (heat shock protein) promoters. Alignment of the predicted protein encoded by ORF-2 showed significant homology to the Bacteroides fragilis tnpA gene from the transposon Tn4555, whereas the ORF-3 protein showed homology to B. fragilis transposase (Tn5220) and integrase (Tn4555) proteins. This suggests a transposition-like event may be responsible for transfer of these genes between Porphyromonas and Prevotella. Received: 8 June 2000 / Accepted: 11 August 2000     

Molecular Characterization and Substrate Preference of a Polycyclic Aromatic Hydrocarbon Dioxygenase from Cycloclasticus sp. Strain A5

Cycloclasticus sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes. The phnA1, phnA2, phnA3, and phnA4 genes, which encode the α and β subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the α and β subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved. Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the E. coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.     

Evidence for sub-families of actin genes in Dictyostelium as determined by comparisons of 3' end sequences

We have sequenced the 3′ end of five actin genomic clones and three actin complementary DNA clones from Dictyostelium. Comparison of the sequences shows that the protein coding regions are highly conserved, while the region corresponding to the 3′ untranslated regions are divergent. Additional analysis indicates regions of homology in the 3′ untranslated region between sets of actin genes. Southern DNA blot hybridization studies using labeled 3′ ends suggest that there are sub-families of actin genes that are related within the 3′ untranslated regions. No homology is found in the sequences outside the messenger RNA encoding regions. Analysis of the sequence data has shown that the difference in length between the ~1.25 × 10³ and ~1.35 × 10³ base actin messenger RNAs is in the lengths of the 3′ untranslated region.     

Evolutionary implications of a complex pattern of DNA sequence homology extending far upstream of the hsp70 genes at loci 87A7 and 87C1 in Drosophila melanogaster

Genes coding for the major 70,000 M_r heat shock protein (hsp70) are found at two loci, 87A7 and 87C1, in Drosophila melanogaster. At 87A7 they are present as two genes in diverging orientation, whilst at 87C1 two tandemly repeated distal copies are separated from a single copy in divergent orientation by about 40,000 bases of DNA. Within this 40,000 bases are found the αβ heat-induced genes, interspersed with γ elements. In this paper we report the isolation and characterization of the proximal hsp70 gene from locus 87C1. The DNA sequence upstream from this gene shows greater than 98% homology with that of αγ, suggesting that the γ element interspersed with αβ sequences originated from this position. In addition, we present the DNA sequence between the two genes in a cloned DNA segment from 87A7, and compare the sequence with those from 87C1. We find a complex pattern of nucleotide sequence homology extending far upstream of the hsp70 genes at the two loci. The evolution of the present arrangement at these two loci is discussed.     

Isolation and characterisation of genes for sulphate activation and reduction in Aspergillus nidulans: implications for evolution of an allosteric control region by gene duplication

A region of the Aspergillus nidulans genome carrying the sA and sC genes, encoding PAPS reductase and ATP sulphurylase, respectively, was isolated by transformation of an sA mutant with a cosmid library. The genes were subcloned and their functions confirmed by retransformation and complementation of A. nidulans strains carrying sA and sC mutations. The physical distance of 2 kb between the genes corresponds to a genetic distance of 1 cM. While the deduced amino acid sequence of the sA gene product shows homology with the equivalent MET16 gene product of Saccharomyces cerevisiae, the sC gene product resembles the equivalent MET3 yeast gene product at the N-terminal end, but differs markedly from it at the C-terminal end, showing homology to the APS kinases of several microorganisms. It is proposed that this C-terminal region does not encode a functional APS kinase, but is responsible for allosteric regulation by PAPS of the sulphate assimilation pathway in A. nidulans, and that the ATP sulphurylase encoding-gene (sC) of filamentous ascomycetes may have evolved from a bifunctional gene similar to the nodQ gene of Rhizobium meliloti.