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 ␣-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.Sinorhizobium meliloti is a gram-negative bacterium that is best known for forming nitrogen-fixing symbiotic relationships with legumes, such as alfalfa (25). When not in symbiosis, these rhizobia are part of the normal, free-living soil microflora. S. meliloti cells detect root exudates in the soil and migrate toward, attach to, and invade the root hairs of alfalfa. This process ultimately leads to the formation of specialized root organs called nodules. During the development of nodules, S. meliloti cells differentiate into endosymbiotic forms called bacteroids, which can reduce N 2 to NH 4 ϩ through an energetically expensive process requiring eight low-potential electrons and at least 16 ATP molecules per molecule of N 2 reduced. The bacteroids provide this fixed nitrogen (NH 4 ϩ ) to the plant, and the plant delivers organic acids and perhaps other carbon and energy sources to the bacteroids (6, 10).The DNA sequence of the S. meliloti strain 1021 genome, which consists of a chromosome (3.65 Mb) and two megaplasmids, pSymA (1.35 Mb) and pSymB (1.68 Mb), has been determined (9). This 6.7-Mb genome was predicted to contain at least 6,207 protein-coding genes along with various insertion sequence elements and phage sequences (9). Having the sequence available is a major step toward describing the biology of the bacteria, but new methods for analyzing the genome are needed in order to obtain a more functional description of the roles played by each of these genetic elements. Manipulating a genome requires extensive use of oligonucleotides, over 12,000 for the S. meliloti genome. The expense of these and the attendant cost of cloning and other standard manipulations are a considerable barrier to working with the entire sequence. However, methods b...

Nitrilotriacetate (NTA), a synthetic chelating agent, has been used for various radionuclide processing and decontamination procedures. NTA has been codisposed with radionuclides and heavy metals to soils and subsurface sediments (4, 27, 32), where it can greatly increase the mobility of these metals in the environment by forming soluble complexes with them. Microbial degradation of NTA may ultimately aid in immobilizing these radionuclides. Several NTA-degrading bacteria have been isolated (2,7,13,18,23,42). An NTA monooxygenase (NTA-Mo) of Chelatobacter heintzii ATCC 29600 has been identified (16) (47). NTA oxidation is proposed to be catalyzed by a heterodimer of components A (cA) and B (cB), but it is unclear how these two components interact with each other or what the function of each component is (44). In order to provide additional information on the function of cA and cB and to facilitate understanding natural attenuation or engineered bioremediation of NTA in the environment through the use of specific PCR primers or gene probes, we cloned, sequenced, and analyzed a gene cluster involved in NTA degradation. On the basis of DNA sequence and activity analysis, the two components were shown to be two separate enzymes, an monooxygenase that oxidized NTA at the expense of reduced flavin mononucleotide (FMNH 2 ) and O 2 and an NADH:flavin mononucleotide (FMN) oxidoreductase that uses NADH to reduce FMN to FMNH 2 .(A preliminary account of this work was presented at the 1995 American Society for Microbiology General Meeting [46], and the DNA sequence and gene organization have been published in a master's degree thesis [46]). MATERIALS AND METHODSBacterial strains and plasmids. The plasmids used or constructed in this study are listed in Table 1. C. heintzii ATCC 29600 was obtained from the American Type Culture Collection (Rockville, Md.) and was cultured with using a mineral salt medium with NTA as the sole carbon source (42). Escherichia coli HB101 was used as the host for pRK311, strain DH5␣ was used as the host for pBS, and strain JM105 was used as the host for pTrc99A. E. coli strains were routinely grown at 37ЊC in Luria-Bertani (LB) medium or on LB agar (37). Tetracycline and ampicillin (Sigma, St. Louis, Mo.) were used at 25 and 100 g/ml, respectively.Gene cloning. The two components of NTA-Mo were purified according to the method of Uetz et al. (44), subjected to discontinuous sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (24), and then electroblotted onto a polyvinylidene difluoride membrane (28, 31) for N-terminal amino acid sequence analysis on an ABI 470 protein sequencer at the Department of Biochemistry and Biophysics, Washington State University.Oligonucleotides were 5Ј end labeled with 32 P by polynucleotide kinase (37). C. heintzii ATCC 29600 genomic DNA was isolated by a combination of largescale CsCl gradient preparation and hexadecyltrimethyl ammonium bromide and phenol extraction (3, 37). The PolarPlex Chemiluminescent Kit of Millipore (Bedford, Mass.) was used to ...

The nitrogen-fixing, symbiotic bacterium Sinorhizobium meliloti reduces molecular dinitrogen to ammonia in a specific symbiotic context, supporting the nitrogen requirements of various forage legumes, including alfalfa. Determining the DNA sequence of the S. meliloti genome was an important step in plant-microbe interaction research, adding to the considerable information already available about this bacterium by suggesting possible functions for many of the >6,200 annotated open reading frames (ORFs). However, the predictive power of bioinformatic analysis is limited, and putting the role of these genes into a biological context will require more definitive functional approaches. We present here a strategy for genetic analysis of S. meliloti on a genomic scale and report the successful implementation of the first step of this strategy by constructing a set of plasmids representing 100% of the 6,317 annotated ORFs cloned into a mobilizable plasmid by using efficient PCR and recombination protocols. By using integrase recombination to insert these ORFs into other plasmids in vitro or in vivo (B. L. House et al., Appl. Environ. Microbiol. 70:2806-2815, 2004), this ORFeome can be used to generate various specialized genetic materials for functional analysis of S. meliloti, such as operon fusions, mutants, and protein expression plasmids. The strategy can be generalized to many other genome projects, and the S. meliloti clones should be useful for investigators wanting an accessible source of cloned genes encoding specific enzymes.

The dicarboxylate transport (Dct) system of Sinorhizobium meliloti, which is essential for a functional nitrogen-fixing symbiosis, has been thought to transport only dicarboxylic acids. We show here that the permease component of the Dct system, DctA, can transport orotate, a monocarboxylic acid, with an apparent K m of 1.7 mM and a V max of 163 nmol min ؊1 per mg of protein in induced cells. DctA was not induced by the presence of orotate. The transport of orotate was inhibited by several compounds, including succinamic acid and succinamide, which are not dicarboxylic acids. The dicarboxylic acid maleate (cis-butenedioic acid) was not an inhibitor of orotate transport, which suggests that it was not recognized by DctA. However, maleate was an excellent inducer of DctA expression. Our evaluation of 17 compounds as inducers and inhibitors of transport suggests that substrates recognized by S. meliloti DctA must have appropriately spaced carbonyl groups and an extended conformation, while good inducers are more likely to have a curved conformation.Soil bacteria belonging to the genera Sinorhizobium, Rhizobium, Bradyrhizobium, and Azorhizobium can form symbiotic associations with leguminous plants. The bacteria elicit the formation of specialized root organs called nodules, in which they reduce atmospheric dinitrogen, and provide the resulting ammonia to the plant. Symbiotic nitrogen fixation requires a large energy input. To provide this energy, the host plant supplies organic compounds such as sucrose, which are transported to the nodules and converted to substrates supplied to the bacteroids (17). The tricarboxylic acid (TCA) cycle intermediates succinate, malate, and fumarate are likely to be the major carbon sources for rhizobial bacteroids in the nodule (20). It is thought that these compounds are imported into the bacteroids using the rhizobial dicarboxylate transport (Dct) system, which in Sinorhizobium meliloti is encoded by three genes located on megaplasmid II, dctA, dctB, and dctD (20). The dctA gene codes for a high-affinity permease. dctA mutants produce nodules that are symbiotically ineffective, and bacteroids from these nodules are unable to transport dicarboxylates (4, 20). The dctB and dctD genes encode a two-component regulatory system, which activates the transcription of dctA in response to the presence of dicarboxylates in the periplasm, where the sensor domain of DctB is located (12, 18). S. meliloti DctA participates in the regulation of its inductiondctA::phoA fusions are induced to a very high level unless there is an active DctA protein in the cell (22).Succinate, malate, fumarate, and aspartate are considered to be substrates for the Dct system (19). Other compounds, including D-lactate, 2-methylsuccinate, 2,2-or 2,3-dimethylsuccinate, acetoacetate, ␤-hydroxybutyrate, mercaptosuccinate, ␣-ketoglutarate, and itaconate, are either substrates or potential substrates for DctA (4, 10, 11). Recently, a study examining fluoroorotic acid (FOA) resistance in Salmonella enterica serovar Typhimuri...

The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to ␤-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput ␤-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.

Resources from the Sinorhizobium meliloti Rm1021 open reading frame (ORF) plasmid libraries were used in a mediumthroughput method to construct a set of 50 overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except the replicon region. Each resulting pSymA derivative carried a defined deletion of approximately 25 ORFs. Various phenotypes, including cytochrome c respiration activity, the ability of the mutants to grow on various carbon and nitrogen sources, and the symbiotic effectiveness of the mutants with alfalfa, were analyzed. This approach allowed us to systematically evaluate the potential impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.

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The gltA gene, encoding Sinorhizobium meliloti 104A14 citrate synthase, was isolated by complementing an Escherichia coli gltA mutant. The S. meliloti gltA gene was mutated by inserting a kanamycin resistance gene and then using homologous recombination to replace the wild-typegltA with the gltA::kanallele. The resulting strain, CSDX1, was a glutamate auxotroph, and enzyme assays confirmed the absence of a requirement for glutamate. CSDX1 did not grow on succinate, malate, aspartate, pyruvate, or glucose. CSDX1 produced an unusual blue fluorescence on medium containing Calcofluor, which is different from the green fluorescence found with 104A14. High concentrations of arabinose (0.4%) or succinate (0.2%) restored the green fluorescence to CSDX1. High-performance liquid chromatography analyses showed that CSDX1 produced partially succinylated succinoglycan. CSDX1 was able to form nodules on alfalfa, but these nodules were not able to fix nitrogen. The symbiotic defect of a citrate synthase mutant could thus be due to disruption of the infection process or to the lack of energy generated by the tricarboxylic acid cycle.