Functional screening of metagenome and genome libraries for detection of novel flavonoid-modifying enzymes.

The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside.

Involvement of multiple loci in quorum quenching of autoinducer I molecules in the nitrogen-fixing symbiont Rhizobium (Sinorhizobium) sp. strain NGR234.

Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. Since we have previously described the complete genome sequence of NGR234, we now report on a genome-wide functional analysis of the genes and enzymes involved in autoinducer I hydrolysis in this microbe. Altogether we identified five cosmid clones that repeatedly gave a positive result in our function-based approach for the detection of autoinducer I hydrolase genes. Of these five cosmid clones, two were located on pNGR234b and three were on cNGR234. Subcloning and in vitro mutagenesis in combination with BLAST analyses identified the corresponding open reading frames (ORFs) of all cosmid clones: dlhR, qsdR1, qsdR2, aldR, and hitR-hydR. Analyses of recombinant DlhR and QsdR1 proteins by using high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrate that these enzymes function as acyl homoserine lactone (AHL) lactonases. Furthermore, we showed that these enzymes inhibited biofilm formation and other quorum-sensing-dependent processes in Pseudomonas aeruginosa, Chromobacterium violaceum, and Agrobacterium tumefaciens. Finally, our experimental data suggest that competitive colonization of roots in the rhizospheres of cowpea plants is affected by DlhR and QsdR1.

Deep amoA amplicon sequencing reveals community partitioning within ammonia-oxidizing bacteria in the environmentally dynamic estuary of the River Elbe.

The community composition of betaproteobacterial ammonia-oxidizing bacteria (ß-AOB) in the River Elbe Estuary was investigated by high throughput sequencing of ammonia monooxygenase subunit A gene (amoA) amplicons. In the course of the seasons surface sediment samples from seven sites along the longitudinal profile of the upper Estuary of the Elbe were investigated. We observed striking shifts of the ß-AOB community composition according to space and time. Members of the Nitrosomonas oligotropha-lineage and the genus Nitrosospira were found to be the dominant ß-AOB within the river transect, investigated. However, continuous shifts of balance between members of both lineages along the longitudinal profile were determined. A noticeable feature was a substantial increase of proportion of Nitrosospira-like sequences in autumn and of sequences affiliated with the Nitrosomonas marina-lineage at downstream sites in spring and summer. Slightly raised relative abundances of sequences affiliated with the Nitrosomonas europaea/Nitrosomonas mobilis-lineage and the Nitrosomonas communis-lineage were found at sampling sites located in the port of Hamburg. Comparisons between environmental parameters and AOB-lineage (ecotype) composition revealed promising clues that processes happening in the fluvial to marine transition zone of the Elbe estuary are reflected by shifts in the relative proportion of ammonia monooxygenase sequence abundance, and hence, we propose ß-AOB as appropriate indicators for environmental dynamics and the ecological condition of the Elbe Estuary.

Novel blue light-sensitive proteins from a metagenomic approach.

A microarray-based approach was used to screen a soil metagenome for the presence of blue light (BL) photoreceptor-encoding genes. The microarray carried 149 different 54-mer oligonucleotides, derived from consensus sequences of light, oxygen and voltage (LOV) domain BL photoreceptor genes. Calibration of the microarrays allowed the detection of minimally 50 ng of genomic DNA against a background of 2-5 microg of genomic DNA. Identification of a positive cosmid clone was still possible for an amount of 0.25 ng against a background of 10 microg of labelled DNA clones. The array could readily identify targets carrying 4% sequence mismatch. Using the LOV microarray, up to 1200 library clones in concentrations of c. 20 ng each with a c. 40 kb insert size could be screened in a single batch. After calibration and reliability controls, the microarray was probed with cosmid-cloned DNA from the thermophilic fraction of a soil sample. From this approach, a novel gene was isolated that encodes a protein consisting of several Per-Arnt-Sim domains, a LOV domain associated to a histidine kinase and a response regulator domain. The novel gene showed highest similarity to a known sequence from Kineococcus radiotolerans SRS30216 (58% identity for the LOV domain only) and to a gene from Methylibium petroleiphilum PM1 (57% identity). The gene, designated as ht-met1 (Hamburg Thermophile Metagenome 1), was isolated and fully sequenced (3615 bp). ht-met1 is followed by a second open reading frame encoding a Fe-chelatase, an arrangement quite frequent for BL photoreceptors. The LOV domain region of ht-met1 was subcloned and expressed yielding a fully functional, flavin-containing LOV domain. Irradiation generated the typical LOV photochemistry, with the transient formation of a flavin-protein photoadduct. The dark recovery lifetime was found as tau(REC) = 120 s (20 degrees C) and is among the fastest ones determined so far for bacterial LOV domains.

Metagenome-derived clones encoding two novel lactonase family proteins involved in biofilm inhibition in Pseudomonas aeruginosa.

Here we report the isolation and characterization of three metagenome-derived clones that interfere with bacterial quorum sensing and degrade N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C(8)-HSL). By using a traI-lacZ gene fusion, the metagenome-derived clones were identified from a soil DNA library and analyzed. The open reading frames linked to the 3-oxo-C(8)-HSL-degrading activities were designated bpiB01, bpiB04, and bpiB07. While the BpiB07 protein was similar to a known lactonase, no significant similarities were observed for the BpiB01 and BpiB04 proteins or the deduced amino acid sequences. High-performance liquid chromatography-mass spectrometry analyses confirmed that the identified genes encode novel lactone-hydrolyzing enzymes. The original metagenome-derived clones were expressed in Pseudomonas aeruginosa and employed in motility and biofilm assays. All clones were able to reproducibly inhibit motility in P. aeruginosa. Furthermore, these genes clearly inhibited biofilm formation in P. aeruginosa when expressed in P. aeruginosa PAO1. Thus, this is the first study in which metagenome-derived proteins have been expressed in P. aeruginosa to successfully inhibit biofilm formation.

Isolation and characterization of a metagenome-derived and cold-active lipase with high stereospecificity for (R)-ibuprofen esters.

We report on the isolation and biochemical characterization of a novel, cold-active and metagenome-derived lipase with a high stereo-selectivity for pharmaceutically important substrates. The respective gene was isolated from a cosmid library derived from oil contaminated soil and designated lipCE. The deduced aa sequence indicates that the protein belongs to the lipase family l.3, with high similarity to Pseudomonas fluorescens lipases containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca(2+)-binding sites. The overexpressed protein revealed a molecular weight of 53.2kDa and was purified by refolding from inclusion bodies after expression in Escherichia coli. The optimum temperature of LipCE was determined to be 30 degrees C. However, the enzyme still displayed 28% residual activity at 0 degrees C and 16% at -5 degrees C. Calcium ions strongly increased activity and thermal stability of the protein. Further detailed biochemical characterization of the recombinant enzyme showed an optimum pH of 7 and that it retained activity in the presence of a range of metal ions and solvents. A detailed analysis of the enzyme's substrate spectrum with more than 34 different substrates indicated that the enzyme was able to hydrolyze a wide variety of substrates including the conversion of long chain fatty acid substrates with maximum activity for pNP-caprate (C(10)). Furthermore LipCE was able to hydrolyze stereo-selectively ibuprofen-pNP ester with a high preference for the (R) enantiomer of >91% ee and it demonstrated selectivity for esters of primary alcohols, whereas esters of secondary or tertiary alcohols were nearly not converted.

Characterization of a metagenome-derived halotolerant cellulase.

Metagenomes of uncultured microorganisms represent a sheer unlimited resource for discovery of novel biocatalysts. Here, we report on the biochemical characterisation of a novel, soil metagenome-derived cellulase (endoglucanase), Cel5A. The deduced amino acid sequence of Cel5A was similar to a family 5, single domain cellulase with no distinct cellulose binding domain from Cellvibrio mixtus. The 1092bp ORF encoding Cel5A was overexpressed in Escherichia coli and the corresponding 42.1 kDa protein purified using three-step chromatography. The recombinant Cel5A protein was highly active against soluble cellulose substrates containing beta-1,4 linkages, such as lichenan and barley beta-glucan, and not active against insoluble cellulose. Glucose was not among the initial hydrolysis products, indicating an endo mode of action. Cel5A displayed a wide range of pH activity with a maximum at pH 6.5 and at least 60% activity at pH 5.5 and 9.0. The enzyme was highly stable at 40 degrees C for up to 11 days, and retained 86-87% activity after incubation with 3M NaCl, 3M RbCl or 4M KCl for 20 h. Cel5A was also active in the presence of diverse divalent cations, detergents and EDTA. This highly stable, salt and pH tolerant cellulase is an ideal candidate for industrial applications.

A biotin-regulated locus, bioS, in a possible survival operon of Rhizobium meliloti.

A novel biotin-inducible DNA locus, bioS, was found in Rhizobium meliloti 1021 by mutation with a transposable promoter probe. The promoterless nptII reporter responded to 40 nM biotin with a sixfold increase in NPTII protein. Stationary-phase mutant cells accumulated 50% more 14C-biotin than corresponding wild-type cells. The prototrophic mutant competed equally with the wild type in biotin-free medium but poorly when 40 nM biotin was present. The 588-bp bioS is located among three genes showing homology to survival operons of other bacteria, and it may be part of a system that R. meliloti uses to respond to plant biotin signals.

Biotin and other water-soluble vitamins are key growth factors for alfalfa root colonization by Rhizobium meliloti 1021.

Rhizosphere growth limitations imposed on Rhizobium meliloti by availability of biotin, thiamine, and riboflavin were overcome by adding nanomolar amounts of these vitamins. Studies done with R. meliloti 1021 showed that both synthesis and uptake of biotin promote colonization of alfalfa roots. Two lines of evidence indicated that plant-derived biotin normally promotes root colonization: (i) adding avidin significantly (P < or = 0.01) reduced rhizosphere growth of R meliloti 1021, and (ii) growth of Tn5-induced biotin auxotrophs still increased 10-fold in the rhizosphere. Synthesis, however, is the more important source of biotin for R. meliloti 1021 because in root colonization tests biotin auxotrophs competed very poorly with the parent strain. Mutations conferring biotin auxotrophy were closely linked on a single restriction fragment, and one was complemented with the Escherichia coli bio operon. Initial nucleotide sequencing and DNA-DNA hybridization tests showed the biotin synthesis genes in R. meliloti are quite different from those in E. coli.

BioS, a biotin-induced, stationary-phase, and possible LysR-type regulator in Sinorhizobium meliloti.

Sinorhizobium meliloti 1021 produces biotin required for growth, but it also responds to external biotin signals from alfalfa plants through the bioS regulatory locus. Mutation of bioS increases biotin uptake, extends stationary phase in the presence of biotin, and impairs competitive growth in the presence of biotin. New data supporting the relevance of this gene to plant-microbe interactions show that a bioS-gusA reporter fusion is expressed by bacteria on plant roots, by bacteria in alfalfa root nodules, and more generally by any stationary-phase bacterial cells in the presence of biotin. Significant transcription of bioS-gusA was detected at 50 pM biotin, and a 16-fold induction was measured in stationary-phase cells with 1 nM biotin. A positive autoregulation of bioS is suggested by the fact that, in the presence of 10 nM biotin, a bioS-gusA fusion in wild-type cells was transcribed at more than twice the rate of the same reporter fusion in a bioS mutant background. Analyses of primer extension products and expression studies with three different bioS-gusA promoter fusions defined major characteristics of the bioS promoter. Expression of bioS in Escherichia coli resulted in the production of a 35-kDa BioS protein with characteristics of an LysR-type regulator. Those traits included a deduced amino acid sequence containing possible helix-turn-helix DNA-binding structures and three T-N(11)-A motifs upstream from the bioS gene. A regulatory role for BioS protein was supported by tests showing that a 321-bp DNA fragment containing the bioS promoter altered the electrophoretic mobility of recombinant BioS and of a corresponding band in protein extracts from S. meliloti 1021. The absence in BioS of any significant homology to known amino acid sequences was consistent with the fact that DNA hybridization tests with seven genera of bacteria detected bioS only in Sinorhizobium.

Sinorhizobium meliloti strain 1021 bioS and bdhA gene transcriptions are both affected by biotin available in defined medium.

Sinorhizobium meliloti 1021 responds to external biotin signals from alfalfa plants through the bioS regulatory locus. Immunogold labeling and electron microscopy revealed that the BioS protein is located within the S. meliloti cytoplasm. Under biotin-limiting conditions the S. meliloti cell lumen was filled with polyhydroxybutyrate (PHB) granules suggesting that either PHB synthesis or degradation are influenced by biotin. To test this hypothesis a 3-hydroxybutyrate-dehydrogenase-lacZ (bdhA-lacZ) fusion was mobilized into S. meliloti. beta-galactosidase tests revealed an overall 3.6-5.2-fold higher bdhA transcription in the presence of added biotin. Comparison of the bdhA and the bioS promoter regions identified several common motifs.

The metagenome-derived enzyme RhaB opens a new subclass of bacterial B type α-L-rhamnosidases.

A combined sequence- and function-based analysis of a metagenomic library DNA derived from elephant feces led to the identification of a novel bacterial α-l-rhamnosidase belonging to glycoside hydrolase family 78 (GH78). The gene was designated rhaB (4095bp) and encoded for a putative protein of 1364 amino acids. The C-terminal part of the enzyme revealed an amino acid (AA) sequence identity of 58% to a predicted bacterial α-l-rhamnosidase from Bacteroides nordii. Interestingly, the N-terminal region of the deduced enzyme RhaB contained a GDSL-like lipase motif and an acetyl-xylan esterase (DAP2) motif. While heterologous expression of the complete rhaB failed, subcloning of the gene identified the most active open reading frame (ORF) to be of 3081bp, which we designated rhaB1. The enzyme RhaB1 was overexpressed in Escherichia coli BL21 (DE3) and was purified to an amount of 75mg/L of culture medium. In accordance to the intestinal origin, RhaB1 showed a preference for mesophilic conditions with an optimum activity at a temperature TOpt of 40°C and a pHOpt of 6.5, respectively. The recombinant protein had a Km value of 0.79mM and a specific activity vmax of 18.4U for pNP-α-l-rhamnose, a calculated Km of 6.36mM and vmax of 2.9×10(-3)U for naringin, and a Km of 6.75mM and specific activity vmax of 8.63×10(-2)U for rutin, respectively. Phylogenetic analysis and amino acid domain architecture comparison revealed that RhaB1 belongs to a new subclass of bacterial B type α-l-rhamnosidases of GH 78. To our knowledge RhaB1 is the first biochemically-characterized enzyme of this subclass.

BpiB05, a novel metagenome-derived hydrolase acting on N-acylhomoserine lactones.

The N-acyl-homoserine lactones (N-AHLs) play an important role in bacterial cell-cell signaling. Up to date, however, only a few different experimentally proven classes of N-AHL ring-cleaving enzymes are known. Here we report on the isolation and biochemical characterization of a novel hydrolase derived from the soil metagenome and acting on N-AHLs. The identified protein designated BpiB05 is weakly similar to hypothetical proteins from Bacteroides fragilis, the draft genomes of two Burkholderia species as well as a marine metagenomic ORF but is otherwise not similar to any known protein. BpiB05 was overexpressed in Escherichia coli as a 10× His-tagged fusion protein. The recombinant protein revealed a molecular weight of about 70kDa and was tested for its quorum quenching (QQ) activities using a lacZ-bioassay. Additional HPLC-MS analyses confirmed the lactonolytic activity of the purified protein in the presence of Ca²âº. Further tests suggested that BpiB05 strongly reduces motility in Pseudomonas aeruginosa, pyocyanin synthesis and biofilm formation in this microbe. Because BpiB05 is not distantly related to any of the currently known hydrolases it forms probably a novel group within the growing number of proteins acting on N-AHLs.

Isolation and biochemical characterization of two novel metagenome-derived esterases.

The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to beta-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50 degrees C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47 degrees C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (+)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications.

Biotin in microbes, the genes involved in its biosynthesis, its biochemical role and perspectives for biotechnological production.

Biotin (vitamin H) is one of the most fascinating cofactors involved in central pathways in pro- and eukaryotic cell metabolism. Since its original discovery in 1901, research has led to the discovery of the complete biotin biosynthesis pathways in many different microbes and much work has been done on the highly intriguing and complex biochemistry of biotin biosynthesis. While humans and animals require several hundred micrograms of biotin per day, most microbes, plants and fungi appear to be able to synthesize the cofactor themselves. Biotin is added to many food, feed and cosmetic products, creating a world market of 10-30 t/year. However, the majority of the biotin sold is synthesized in a chemical process. Since the chemical synthesis is linked with a high environmental burden, much effort has been put into the development of biotin-overproducing microbes. A summary of biotin biosynthesis and its biological role is presented; and current strategies for the improvement of microbial biotin production using modern biotechnological techniques are discussed.

Recombinant Rhizobium meliloti strains with extra biotin synthesis capability.

The growth of Rhizobium meliloti 1021 in an experimental alfalfa (Medicago sativa L.) rhizosphere was stimulated by adding nanomolar amounts of biotin. To overcome this biotin limitation, R. meliloti strains were constructed by conjugating the Escherichia coli biotin synthesis operon into biotin auxotroph R. meliloti 1021-B3. Transconjugant strains Rm1021-WS10 and Rm1021-WS11 grew faster in vitro and achieved a higher cell density than did R. meliloti 1021 and overproduced biotin on a defined medium. The increase in cell yield was associated with as much as a 99% loss in viability for Rm1021-WS11, but data suggested that a separate stabilizing factor in the E. coli DNA reduced cell death in Rm1021-WS10. In rhizosphere tests, the recombinant strains showed delayed growth and competed poorly against Rm1021.

Molecular characterization of the Sinorhizobium meliloti nlpD gene.

The Sinorhizobium meliloti nlpD gene consists of 1,539 nucleotides and codes for 512 amino acids. Expression of the nlpD gene as a histidine-tagged protein in Escherichia coli resulted in the production of a 57-kDa protein. The deduced polypeptide sequence of NlpD contains one unusual hexamer repeat (KVQRGQ), one tetramer (TVTV) and two direct and inverted trimer repeats (KAA, AAK). The N-terminal amino acid residues displayed similarity with signal peptides of secreted bacterial lipoproteins. Mutations of the S. meliloti nlpD gene caused decreased survival of cells in the stationary phase.

Prospecting for novel biocatalysts in a soil metagenome.

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an alpha-amylase (amyA), a 1,4-alpha-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.

Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia.

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).

An evolutionary hot spot: the pNGR234b replicon of Rhizobium sp. strain NGR234.

Rhizobium sp. strain NGR234 has an exceptionally broad host range and is able to nodulate more than 112 genera of legumes. Since the overall organization of the NGR234 genome is strikingly similar to that of the narrow-host-range symbiont Rhizobium meliloti strain 1021 (also known as Sinorhizobium meliloti), the obvious question is why are the spectra of hosts so different? Study of the early symbiotic genes of both bacteria (carried by the SymA plasmids) did not provide obvious answers. Yet, both rhizobia also possess second megaplasmids that bear, among many other genes, those that are involved in the synthesis of extracellular polysaccharides (EPSs). EPSs are involved in fine-tuning symbiotic interactions and thus may help answer the broad- versus narrow-host-range question. Accordingly, we sequenced two fragments (total, 594 kb) that encode 575 open reading frames (ORFs). Comparisons revealed 19 conserved gene clusters with high similarity to R. meliloti, suggesting that a minimum of 28% (158 ORFs) of the genetic information may have been acquired from a common ancestor. The largest conserved cluster carried the exo and exs genes and contained 31 ORFs. In addition, nine highly conserved regions with high similarity to Agrobacterium tumefaciens C58, Bradyrhizobium japonicum USDA110, and Mesorhizobium loti strain MAFF303099, as well as two conserved clusters that are highly homologous to similar regions in the plant pathogen Erwinia carotovora, were identified. Altogether, these findings suggest that >/==" BORDER="0">40% of the pNGR234b genes are not strain specific and were probably acquired from a wide variety of other microbes. The presence of 26 ORFs coding for transposases and site-specific integrases supports this contention. Surprisingly, several genes involved in the degradation of aromatic carbon sources and genes coding for a type IV pilus were also found.