Regulation of succinoglycan and galactoglucan biosynthesis in Sinorhizobium meliloti

Sinorhizobium meliloti (Rhizobium meliloti) 2011 has the ability to produce the two acidic exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II). EPS I is a branched heteropolysaccharide composed of octasaccharide repeating units, whereas EPS II is a linear heteropolysaccharide consisting of disaccharide subunits. The exo-exs and exp gene clusters are involved in the biosynthesis of EPSI and EPSII, respectively. EPSI and EPSII biosynthesis genes are differentially expressed resulting in a complex regulation of EPS production in S. meliloti. The phosphate concentration was identified as an important factor affecting the expression of exp genes.     

Responses of the model legume Medicago truncatula to the rhizobial exopolysaccharide succinoglycan

Many species of rhizobial bacteria can invade their plant hosts and induce development of symbiotic nitrogen-fixing nodules only if they are able to produce an acidic exopolysaccharide (EPS) with certain structural and molecular weight characteristics.^1–3 Sinorhizobium meliloti that produces the functional form of the exopolysaccharide succinoglycan induces formation of invasion structures called infection threads in the root hair cells of its plant hosts alfalfa and Medicago truncatula. However, S. meliloti mutants that cannot produce succinoglycan are not able to induce infection thread formation, resulting in an early arrest of nodule development and in nitrogen starvation of the plant. Mounting evidence has suggested that succinoglycan acts as a signal to these host plants to permit the entry of S. meliloti. Now, our microarray screen and functional category analysis of differentially-expressed genes show that M. truncatula plants inoculated with wild type S. meliloti receive a signal to increase their translation capacity, alter their metabolic activity and prepare for invasion, while those inoculated with a succinoglycan-deficient mutant do not receive this signal, and also more strongly express plant defense genes.Key words: nitrogen fixation, nodule, succinoglycan, microarray, legume, rhizobial bacteria, Sinorhizobium meliloti, Medicago truncatula, infection thread, root hair     

Regulation of motility by the ExpR/Sin quorum-sensing system in Sinorhizobium meliloti

A successful symbiotic relationship between Sinorhizobium meliloti and its host Medicago sativa (alfalfa) depends on several signaling mechanisms, such as the biosynthesis of exopolysaccharides (EPS) by S. meliloti. Previous work in our laboratory has shown that a quorum-sensing mechanism controls the production of the symbiotically active EPS II. Recent microarray analysis of the whole-genome expression profile of S. meliloti reveals that the ExpR/Sin quorum-sensing system regulates additional physiological processes that include low-molecular-weight succinoglycan production, nitrogen utilization, metal transport, motility, and chemotaxis. Nearly half of the flagellar genes and their dependence on quorum sensing are prominently displayed in our microarray analyses. We extend those observations in this work and confirm the findings by real-time PCR expression analysis of selected genes, including the flaF, flbT, flaC, cheY1, and flgB genes, involved in motility and chemotaxis. These genes code for regulators of flagellum synthesis, the chemotactic response, or parts of the flagellar apparatus. Gene expression analyses and visualization of flagella by electron microscopy performed at different points in the growth phase support our proposed model in which quorum sensing downregulates motility in S. meliloti. We demonstrate that the ExpR/Sin quorum-sensing system controls motility gene expression through the VisN/VisR/Rem relay. We also show that the ExoS-dependent two-component system suppresses motility gene expression through VisN and Rem in parallel to quorum sensing. This study contributes to our understanding of the mechanisms that govern motility in S. meliloti.     

Analysis of the Rhizobium meliloti exoH/exoK/exoL fragment: ExoK shows homology to excreted endo-β-1,3-1,4-glucanases and ExoH resembles membrane proteins

Nucleotide sequencing of a 4.15 kb DNA fragment from megaplasmid 2 of Rhizobium meliloti 2011 revealed the location of the genes exoH, exoK and exoL. The putative proteins encoded by these genes have molecular weights of 41, 30, and 44 kDa, respectively. The hydrophobicity profile of the ExoH amino acid sequence resembles that of transmembrane proteins. The predicted exoL gene product does not contain hydrophobic regions, indicating a cytoplasmic localization. The exoK gene product is characterized by a putative signal peptide and exhibits significant homology to endo-β-1,3 1,4-glucanases of bacilli and Clostridium thermocellum. R. meliloti exoK mutants induced pink nodules and synthesized a reduced amount of exopolysaccharide (EPS). Colonies of this mutant showed a delay in the appearance of the Calcofluor white fluorescence. In addition, the formation of the characteristic halo was strongly delayed. R. meliloti exoL and exoH mutants induced pseudonodules. The exoH, but not the exoL mutant, synthesized an EPS that could be precipitated by cetyl pyridinium chloride (CPC) and also by ethanol. Plasmid integration mutagenesis revealed promoter regions preceding exoH, exoK and exoL.     

EPS II-Dependent Autoaggregation of Sinorhizobium meliloti Planktonic Cells

Planktonic cells of Sinorhizobium meliloti, a Gram-negative symbiotic bacterium, display autoaggregation under static conditions. ExpR is a LuxR-type regulator that controls many functions in S. meliloti, including synthesis of two exopolysaccharides, EPS I (succinoglycan) and EPS II (galactoglucan). Since exopolysaccharides are important for bacterial attachment, we studied the involvement of EPS I and II in autoaggregation of S. meliloti. Presence of an intact copy of the expR locus was shown to be necessary for autoaggregation. A mutant incapable of producing EPS I displayed autoaggregation percentage similar to that of parental strain, whereas autoaggregation was significantly lower for a mutant defective in biosynthesis of EPS II. Our findings clearly indicate that EPS II is the essential component involved in autoaggregation of planktonic S. meliloti cells, and that EPS I plays no role in such aggregation.     

Diversity and evolution of polyketide biosynthesis gene clusters in the Ceratocystidaceae

Polyketides are secondary metabolites with diverse biological activities. Polyketide synthases (PKS) are often encoded from genes clustered in the same genomic region. Functional analyses and genomic studies show that most fungi are capable of producing a repertoire of polyketides. We considered the potential of Ceratocystidaceae for producing polyketides using a comparative genomics approach. Our aims were to identify the putative polyketide biosynthesis gene clusters, to characterize them and predict the types of polyketide compounds they might produce. We used sequences from nineteen species in the genera, Ceratocystis, Endoconidiophora, Davidsoniella, Huntiella, Thielaviopsis and Bretziella, to identify and characterize PKS gene clusters, by employing a range of bioinformatics and phylogenetic tools. We showed that the genomes contained putative clusters containing a non-reducing type I PKS and a type III PKS. Phylogenetic analyses suggested that these genes were already present in the ancestor of the Ceratocystidaceae. By contrast, the various reducing type I PKS-containing clusters identified in these genomes appeared to have distinct evolutionary origins. Although one of the identified clusters potentially allows for the production of melanin, their functional characterization will undoubtedly reveal many novel and important compounds implicated in the biology of the Ceratocystidaceae.     

Genetic characterization of a mutant of Sinorhizobium fredii strain USDA208 with enhanced competitive ability for nodulation of soybean, Glycine max (L.) Merr.

Sinorhizobium fredii strain USDA208 is a nitrogen-fixing bacterium that forms nodules on roots of soybean and other legume plants. We previously found that the Tn5-containing mutant 208T3, which was derived from strain USDA208, is both deficient in production of exopolysaccharides and more competitive than the wild-type strain in competing against other rhizobia for nodulation of soybean. We now demonstrate that the transposon insertion of the mutant lies in a locus that is highly homologous to a portion of the exo region, which functions in exopolysaccharide biosynthesis by Sinorhizobium meliloti. We sequenced 2906 bp surrounding the insertion site and identified three genes: exoA, exoM, and exoO. The transposon lies within exoM, a glucosyl transferase. A cosmid containing exoHKLAMONP of S. meliloti restores exopolysaccharide production by mutant 208T3 to wild-type levels. Although exo mutants of S. meliloti are defective in their abilities to form indeterminate nodules, the capacities of mutant 208T3 and its wild-type parent to form such nodules on five legume species are indistinguishable. Thus the symbiotic function of exopolysaccharide in S. fredii appears to differ fundamentally from that in S. meliloti.     

Genome-wide comparison of genes involved in the biosynthesis,metabolism, and signaling of juvenile hormone between silkworm and other insects

Juvenile hormone (JH) contributes to the regulation of larval molting and metamorphosis in insects. Herein, we comprehensively identified 55 genes involved in JH biosynthesis, metabolism and signaling in the silkworm (Bombyx mori) as well as 35 in Drosophila melanogaster, 35 in Anopheles gambiae, 36 in Apis mellifera, 47 in Tribolium castaneum, and 44 in Danaus plexippus. Comparative analysis showed that each gene involved in the early steps of the mevalonate (MVA) pathway, in the neuropeptide regulation of JH biosynthesis, or in JH signaling is a single copy in B. mori and other surveyed insects, indicating that these JH-related pathways or steps are likely conserved in all surveyed insects. However, each gene participating in the isoprenoid branch of JH biosynthesis and JH metabolism, together with the FPPS genes for catalyzing the final step of the MVA pathway of JH biosynthesis, exhibited an obvious duplication in Lepidoptera, including B. mori and D. plexippus. Microarray and real-time RT-PCR analysis revealed that different copies of several JH-related genes presented expression changes that correlated with the dynamics of JH titer during larval growth and metamorphosis. Taken together, the findings suggest that duplication-derived copy variation of JH-related genes might be evolutionarily associated with the variation of JH types between Lepidoptera and other insect orders. In conclusion, our results provide useful clues for further functional analysis of JH-related genes in B. mori and other insects.     

Plant defence and delayed infection of alfalfa pseudonodules induced by an exopolysaccharide (EPS I)-deficient Rhizobium meliloti mutant

Mutants of the symbiotic soil bacterium Rhizobium meliloti that fail to synthesize the acidic exopolysaccharide EPS I were unable to induce infected root nodules on Medicago sativa L. (alfalfa). These strains, however, elicited pseudonodules that contained no infection threads or bacteroids. The cortical cell walls of the pseudonodules were abnormally thick and incrusted with an autofluorescent material. Parts of these cell walls and wall appositions contained callose. Biochemical analysis of nodules induced by the EPS I-deficient R. meliloti mutant revealed an increase of phenolic compounds bound to the nodule cell walls when compared with the wild-type strain. These microscopic and biochemical data indicated that a general plant defence response against the EPS I-deficient mutant of R. meliloti was induced in alfalfa pseudonodules. Following prolonged incubation with the EPS I-deficient R. meliloti mutant, the defence system of the alfalfa plant could be overcome by the rhizobium mutant. In the case of the delayed infections, the mutants colonized lobes of the pseudonodules, but the infection threads in these nodules had an abnormal morphology. They were greatly enlarged and did not contain the typical gum-like matrix inside. The bacteria were tightly packed. Based on the mechanism of phytopathogenic interactions, we propose that EPS I or a related compound may act as a suppressor of the alfalfa plant defence system, enabling R. meliloti to infect the plant.     

Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534

The Gram-negative bacterium Flavobacterium sp. strain R1534 is a natural producer of zeaxanthin. A 14 kb genomic DNA fragment of this organism has been cloned and a 5.1 kb piece containing the carotenoid biosynthesis genes sequenced. The carotenoid biosynthesis cluster consists of five genes arranged in at least two operons. The five genes are necessary and sufficient for the synthesis of zeaxanthin. The encoded proteins have significant homology to the crtE, crtB, crtY, crtI and crtZ gene products of other carotenogenic organisms. Biochemical assignment of the individual gene products was done by HPLC analysis of the carotenoid accumulation in Escherichia coli host strains transformed with plasmids carrying deletions of the Flavobacterium sp. strain R1534 carotenoid biosynthesis cluster.     

Functional characterization of the genes tauO, tauK, and tauI in the biosynthesis of tautomycetin

Tautomycetin is a specific protein phosphatase I inhibitor. In an effort to elucidate the biosynthetic mechanism of tautomycetin, we inactivated genes of the tautomycetin biosynthetic gene cluster, tauI, tauO, and tauK, which encode for putative P450 oxidase, citryl-CoA lyase, and esterase enzymes, respectively. The mutant STQ0606 (??tauO) did not produce any detectable amount of tautomycetin intermediates but could convert dialkylmaleic anhydride to tautomycetin, strongly indicating that TauO was involved in dialkylmaleic anhydride biosynthesis. STQ1211 (??tauK) accumulated dialkylmaleic anhydride, whereas the cofermentation of STQ1211 (??tauK) and STQ0606 (??tauO) restored the production of tautomycetin. Together, these results suggest that TauK was responsible for the conjugation of dialkylmaleic anhydride and the polyketide moiety in tautomycetin biosynthesis. The disruption of tauI resulted in the accumulation of 5-des-keto-tautomycetin, revealing that TauI was responsible for the oxidation at C5 as the last step. Although the shunt pathways were involved in the biosynthesis of tautomycetin, the main post-polyketide synthase tailoring steps were dehydration, decarboxylation and oxidation, taking place consecutively. This study allowed us to predict the biosynthesis of tautomycetin more accurately and provided novel insights into the mechanism of the biosynthesis of tautomycetin.     

Interspecies regulation of the recA gene of gram-negative bacteria lacking an E. coli-like SOS operator

The recA genes of Agrobacterium tumefaciens, Rhizobium meliloti, Rhizobium phaseoli and Rhodobacter sphaeroides, species belonging to the alpha-group bacteria of the Proteobacteria class, have been fused in vitro to the lacZ gene of Escherichia coli. By using a mini-Tn5 transposon derivative, each of these recA-lacZ fusions was introduced into the chromosome of each of the four species, and into that of E. coli. The recA genes of three of the alpha bacteria are induced by DNA damage when inserted in A. tumefaciens, R. phaseoli or R. meliloti chromosomes. The expression of the recA gene of R. sphaeroides is DNA damage-mediated only when present in its own chromosome; none of the genes is induced in E. coli. Likewise, the recA gene of E. coli is not induced in any of the four alpha species. These data indicate that A. tumefaciens, R. meliloti and R. phaseoli possess a LexA-like repressor, which is able to block the expression of their recA genes, as well as that of R. sphaeroides, but not the recA gene of E. coli. The LexA repressor of R. sphaeroides does not repress the recA gene of A. tumefaciens, R. meliloti, R. phaseoli or E. coli.