Novel genes related to nodulation, secretion systems, and surface structures revealed by a genome draft of Rhizobium tropici strain PRF 81

Rhizobium tropici is representative of the diversity of tropical rhizobia, besides comprising strains very effective in fixing N₂ in symbiosis with the common bean (Phaseolus vulgaris L.). The genome of a Brazilian commercial inoculant R. tropici strain (PRF 81, =SEMIA 4088), estimated at 7.85 Mb, was analyzed through a total of 9,026 shotgun reads, assembled in 1,668 phrap contigs, and covering ≈30% of the genome. Annotation identified 2,135 coding DNA sequences (CDS), and only 57.2% have possible functions. The genome comprises a mosaic of genes, with CDS showing the highest similarities with 134 microorganisms, none of which represents more than 19% of the CDS with putative known functions. The high saprophytic capacity of PRF 81 may reside in a variety of genes related to transport, biodegradation of xenobiotics, defense, and secretion proteins, many of which were reported for the first time in the present study. Novelty was also found in nodulation (nodG, a double nodIJ system, nodT, nolF, nolG) and capsular polysaccharide genes, showing stronger similarities with Sinorhizobium (=Ensifer) than with the main symbionts of the common bean—R. etli and R. leguminosarum—suggesting that the original host of R. tropici might be another tropical legume or emphasizing the highly promiscuous nature of this rhizobial species.     

Kinetics study of pyridine biodegradation by a novel bacterial strain,Rhizobium sp. NJUST18

Biodegradation of pyridine by a novel bacterial strain, Rhizobium sp. NJUST18, was studied in batch experiments over a wide concentration range (from 100 to 1,000 mg l^?1). Pyridine inhibited both growth of Rhizobium sp. NJUST18 and biodegradation of pyridine. The Haldane model could be fitted to the growth kinetics data well with the kinetic constants μ* = 0.1473 h^?1, K _s = 793.97 mg l^?1, K _i = 268.60 mg l^?1 and S _m = 461.80 mg l^?1. The true μ _max, calculated from μ*, was found to be 0.0332 h^?1. Yield coefficient Y _X/S depended on S _i and reached a maximum of 0.51 g g^?1 at S _i of 600 mg l^?1. V _max was calculated by fitting the pyridine consumption data with the Gompertz model. V _max increased with initial pyridine concentration up to 14.809 mg l^?1 h^?1. The q _S values, calculated from $V_{ \hbox{max} }$ , were fitted with the Haldane equation, yielding q _Smax = 0.1212 g g^?1 h^?1 and q* = 0.3874 g g^?1 h^?1 at S _m′ = 507.83 mg l^?1, K _s′ = 558.03 mg l^?1, and K _i′ = 462.15 mg l^?1. Inhibition constants for growth and degradation rate value were in the same range. Compared with other pyridine degraders, μ _max and S _m obtained for Rhizobium sp. NJUST18 were relatively high. High K _i and K _i′ values and extremely high K _s and K _s′ values indicated that NJUST18 was able to grow on pyridine within a wide concentration range, especially at relatively high concentrations.     

lpsZ, a lipopolysaccharide gene involved in symbiosis of Rhizobium meliloti.

lpsZ+ is an allele that allows exo (exopolysaccharide-deficient) mutants of Rhizobium meliloti to invade nodules by modifying rhizobial lipopolysaccharide. We have cloned and sequenced the lpsZ gene. The predicted LpsZ protein has a molecular weight of 48,589 and is probably localized in the cytoplasm. A beta-glucuronidase fusion in the lpsZ gene indicates that lpsZ is not regulated by oxygen or nitrogen.     

NopB, a type III secreted protein of Rhizobium sp. strain NGR234, is associated with pilus-like surface appendages

Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGROmegarhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.     

Characterization of NopP, a type III secreted effector of Rhizobium sp. strain NGR234

The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid- and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect.     

Clustered genes required for the synthesis of heterocyst envelope polysaccharide in Anabaena sp. strain PCC 7120

As demonstrated with alr2835 (hepA) and alr2834 (hepC) mutants, heterocysts of Anabaena sp. strain PCC 7120, a filamentous cyanobacterium, must have an envelope polysaccharide layer (the Hep+ phenotype) to fix dinitrogen in an oxygen-containing milieu (the Fox+ phenotype). Transpositions presumptively responsible for a Fox- phenotype were localized in open reading frames (ORFs) near hepA and hepC. A mutation in each of nine of these ORFs was complemented by a clone bearing only that single, intact ORF. Heterocysts of the nine mutants were found to lack an envelope polysaccharide layer. Complementation of mutations in alr2832 and alr2840 may have resulted from recombination. However, alr2825, alr2827, alr2831, alr2833, alr2837, alr2839, and alr2841, like hepA and hepC, are required for a Hep+ Fox+ phenotype.     

Role of BacA in lipopolysaccharide synthesis, peptide transport, and nodulation by Rhizobium sp. strain NGR234

BacA of Sinorhizobium meliloti plays an essential role in the establishment of nitrogen-fixing symbioses with Medicago plants, where it is involved in peptide import and in the addition of very-long-chain fatty acids (VLCFA) to lipid A of lipopolysaccharide (LPS). We investigated the role of BacA in Rhizobium species strain NGR234 by mutating the bacA gene. In the NGR234 bacA mutant, peptide import was impaired, but no effect on VLCFA addition was observed. More importantly, the symbiotic ability of the mutant was comparable to that of the wild type for a variety of legume species. Concurrently, an acpXL mutant of NGR234 was created and assayed. In rhizobia, AcpXL is a dedicated acyl carrier protein necessary for the addition of VLCFA to lipid A. LPS extracted from the NGR234 mutant lacked VLCFA, and this mutant was severely impaired in the ability to form functional nodules with the majority of legumes tested. Our work demonstrates the importance of VLCFA in the NGR234-legume symbiosis and also shows that the necessity of BacA for bacteroid differentiation is restricted to specific legume-Rhizobium interactions.     

Two novel decarboxylase genes play a key role in the stereospecific catabolism of dehydrodiconiferyl alcohol in Sphingobium sp. strain SYK‐6

Sphingobium sp. strain SYK‐6 is able to use a phenylcoumaran‐type biaryl, dehydrodiconiferyl alcohol (DCA), as a sole source of carbon and energy. In SYK‐6 cells, the alcohol group of the B‐ring side chain of DCA was first oxidized to the carboxyl group, and then the alcohol group of the A‐ring side chain was oxidized to generate 5‐(2‐carboxyvinyl)‐2‐(4‐hydroxy‐3‐methoxyphenyl)‐7‐methoxy‐2,3‐dihydrobenzofuran‐3‐carboxylate (DCA‐CC). We identified phcF, phcG and phcH, which conferred the ability to convert DCA‐CC into 3‐(4‐hydroxy‐3‐(4‐hydroxy‐3‐methoxystyryl)‐5‐methoxyphenyl)acrylate (DCA‐S) in a host strain. These genes exhibited no significant sequence similarity with known enzyme genes, whereas phcF and phcG, which contain a DUF3237 domain of unknown function, showed 32% amino acid sequence identity with each other. The DCA‐CC conversion activities were markedly decreased by disruption of phcF and phcG, indicating that phcF and phcG play dominant roles in the conversion of DCA‐CC. Purified PhcF and PhcG catalysed the decarboxylation of the A‐ring side chain of DCA‐CC, producing DCA‐S, and showed enantiospecificity towards (+)‐ and (–)‐DCA‐CC respectively. PhcF and PhcG formed homotrimers, and their K_m for DCA‐CC were determined to be 84 μM and 103 μM, and V_max were 307 μmol?min^?1?mg^?1 and 137 μmol?min^?1?mg^?1 respectively. In conclusion, PhcF and PhcG are enantiospecific decarboxylases involved in phenylcoumaran catabolism.     

Rhodococcus sp. strain TM1 plays a synergistic role in the degradation of piperidine by Mycobacterium sp. strain THO100

Mycobacterium sp. strain THO100 and Rhodococcus sp. strain TM1 were isolated from a morpholine-containing enrichment culture of activated sewage sludge. Strain THO100, but not strain TM1, was able to degrade alicyclic amines such as morpholine, piperidine, and pyrrolidine. The mixed strains THO100 and TM1 showed a better growth on piperidine as the substrate than the pure strain THO100 because strain TM1 was able to reduce the level of glutaraldehyde (GA) produced during piperidine degradation. GA was toxic to strain THO100 (IC₅₀ = 28.3 μM) but less toxic to strain TM1 (IC₅₀ = 215 μM). Strain THO100 possessed constitutive semialdehyde dehydrogenases, namely Sad1 and Sad2, whose activities toward succinic semialdehyde (SSA) were strongly inhibited by GA. The two isozymes were identified as catalase–peroxidase (KatG = Sad1) and semialdehyde dehydrogenase (Sad2) based on mass spectrometric analyses of tryptic peptides and database searches of the partial DNA sequences of their genes. In contrast, strain TM1 containing another constitutive enzyme Gad1 could oxidize both SSA and GA. This study suggested that strain TM1 possessing Gad1 played a synergistic role in reducing the toxic and inhibitory effects of GA produced in the degradation of piperidine by strain THO100.     

Group 3 sigma factor gene, sigJ, a key regulator of desiccation tolerance, regulates the synthesis of extracellular polysaccharide in cyanobacterium Anabaena sp. strain PCC 7120.

The changes in the expression of sigma factor genes during dehydration in terrestrial Nostoc HK-01 and aquatic Anabaena PCC 7120 were determined. The expression of the sigJ gene in terrestrial Nostoc HK-01, which is homologous to sigJ (alr0277) in aquatic Anabaena PCC 7120, was significantly induced in the mid-stage of dehydration. We constructed a higher-expressing transformant of the sigJ gene (HE0277) in Anabaena PCC 7120, and the transformant acquired desiccation tolerance. The results of Anabaena oligonucleotide microarray experiments showed that a comparatively large number of genes relating to polysaccharide biosynthesis were upregulated in the HE0277 cells. The extracellular polysaccharide released into the culture medium of the HE0277 cells was as much as 3.2-fold more than that released by the control cells. This strongly suggests that the group 3 sigma factor gene sigJ is fundamental and conducive to desiccation tolerance in these cyanobacteria.     

NopP, a phosphorylated effector of Rhizobium sp. strain NGR234, is a major determinant of nodulation of the tropical legumes Flemingia congesta and Tephrosia vogelii

Rhizobium sp. NGR234 nodulates many plants, some of which react to proteins secreted via a type three secretion system (T3SS) in a positive- (Flemingia congesta, Tephrosia vogelii) or negative- (Crotalaria juncea, Pachyrhizus tuberosus) manner. T3SSs are devices that Gram-negative bacteria use to inject effector proteins into the cytoplasm of eukaryotic cells. The only two rhizobial T3SS effector proteins characterized to date are NopL and NopP of NGR234. NopL can be phosphorylated by plant kinases and we show this to be true for NopP as well. Mutation of nopP leads to a dramatic reduction in nodule numbers on F. congesta and T. vogelii. Concomitant mutation of nopL and nopP further diminishes nodulation capacity to levels that, on T. vogelii, are lower than those produced by the T3SS null mutant NGR(Omega)rhcN. We also show that the T3SS of NGR234 secretes at least one additional effector, which remains to be identified. In other words, NGR234 secretes a cocktail of effectors, some of which have positive effects on nodulation of certain plants while others are perceived negatively and block nodulation. NopL and NopP are two components of this mix that extend the ability of NGR234 to nodulate certain legumes.     

Draft genome sequence of Rhizobium sp. strain PDO1-076, a bacterium isolated from Populus deltoides

Rhizobium sp. strain PDO1-076 is a plant-associated bacterium isolated from Populus deltoides, and its draft genome sequence is reported.     

Identification of the key genes involved in the degradation of homocholine by Pseudomonas sp. strain A9 by using suppression subtractive hybridization

Microbial transformation of homocholine plays a central role in many biological systems and influence on all kingdoms of life. Here, we used suppression subtractive hybridization (SSH) approach to screen for genes that differentially expressed in response to homocholine by Pseudomonas sp. strain A9 and to gain deep acknowledge about the gene expression and sequences of homocholine degrading enzymes. Twenty-seven differentially expressed genes were identified and were found to involve in the uptake and metabolism of homocholine as well as physiological responses of strain A9 to this compound. Of them, fragments of homocholine dehydrogenase (hcdH), β-alanine betaine aldehyde dehydrogenase (bABALDH), β-alaninebetaine CoA transferase (hcdD), 3-hydroxypropionate dehydrogenase (hcdB), and malonate semialdehyde dehydrogenase (hcdC) genes were detected. After excessive experiments of PCR and sequencing, the full-length sequences of these key genes were identified. Interestingly, a complete sequence of a unique gene cluster (6.2 kbp) of hcd (homocholine degrading) genes that contain the genes hcdD, hcdB, hcdC, and hcdR was obtained. The sequence information of these essential genes will enhance our understanding of homocholine catabolic pathway in microorganisms and will help in identifying better inhibitors or activators of these enzymes to either improve or suppress their activity depending on the importance of the formed metabolite.     

Structure and evolution of NGRRS-1, a complex, repeated element in the genome of Rhizobium sp. strain NGR234.

Much of the remarkable ability of Rhizobium sp. strain NGR234 to nodulate at least 110 genera of legumes, as well as the nonlegume Parasponia andersonii, stems from the more than 80 different Nod factors it secretes. Except for nodE, nodG, and nodPQ, which are on the chromosome, most Nod factor biosynthesis genes are dispersed over the 536,165-bp symbiotic plasmid, pNGR234a. Mosaic sequences and insertion sequences (ISs) comprise 18% of pNGR234a. Many of them are clustered, and these IS islands divide the replicon into large blocks of functionally related genes. At 6 kb, NGRRS-1 is a striking example: there is one copy on pNGR234a and three others on the chromosome. DNA sequence comparisons of two NGRRS-1 elements identified three types of IS, NGRIS-2, NGRIS-4, and NGRIS-10. Here we show that all four copies of NGRRS-1 probably originated from transposition of NGRIS-4 into a more ancient IS-like sequence, NGRIS-10. Remarkably, all nine copies of NGRIS-4 have transposed into other ISs. It is unclear whether the accumulation of potentially mutagenic sequences in large clusters is due to the nature of the IS involved or to some selection process. Nevertheless, a direct consequence of the preferential targeting of transposons into such IS islands is to minimize the likelihood of disrupting vital functions.     

Sequence and analysis of the rpoN sigma factor gene of rhizobium sp. strain NGR234, a primary coregulator of symbiosis.

We report the nucleotide sequence of the rpoN gene from broad-host-range Rhizobium sp. strain NGR234 and analyze the encoded RPON protein, a sigma factor. Comparative analysis of the deduced amino acid sequence of RPON from NGR234 with sequences from other gram-negative bacteria identified a perfectly conserved RPON box unique to RPON sigma factors. Symbiotic regulatory phenotypes were defined for a site-directed internal deletion within the coding sequence of the rpoN gene of Rhizobium strain NGR234: they included quantitative nodulation kinetics on Vigna unguiculata and microscopic analysis of the Fix- determinate nodules of V. unguiculata and Macroptilium atropurpureum. RPON was a primary coregulator of nodulation and was implicated in establishment or maintenance of the plant-synthesized peribacteroid membrane. Phenotypes of rpoN in Rhizobium strain NGR234 could be grouped as symbiosis related, rather than simply pleiotropically physiological as in free-living bacteria such as Klebsiella pneumoniae and Pseudomonas putida.     

pyd genes of Rhizobium sp. strain TAL1145 are required for degradation of 3-hydroxy-4-pyridone, an aromatic intermediate in mimosine metabolism

Rhizobium sp. strain TAL1145 degrades the Leucaena toxin mimosine and its degradation product 3-hydroxy-4-pyridone (HP). The aim of this investigation is to characterize the Rhizobium genes for HP degradation and transport. These genes were localized by subcloning and mutagenesis on a previously isolated cosmid, pUHR263, containing mid genes of TAL1145 required for mimosine degradation. Two structural genes, pydA and pydB, encoding a metacleavage dioxygenase and a hydrolase, respectively, are required for degradation of HP, and three genes, pydC, pydD, and pydE, encoding proteins of an ABC transporter, are involved in the uptake of HP by TAL1145. These genes are induced by HP, although both pydA and pydB show low levels of expression without HP. pydA and pydB are cotranscribed, while pydC, pydD, and pydE are each transcribed from separate promoters. PydA and PydB show no homology with other dioxygenases and hydrolases in Sinorhizobium meliloti, Mesorhizobium loti, and Bradyrhizobium japonicum. Among various root nodule bacteria, the ability to degrade mimosine or HP is unique to some Leucaena-nodulating Rhizobium strains.     

Sulphation of Rhizobium sp. NGR234 Nod factors is dependent on noeE, a new host-specificity gene

Rhizobia secrete specific lipo-chitooligosaccharide signals (LCOs) called Nod factors that are required for infection and nodulation of legumes. In Rhizobium sp. NGR234, the reducing N -acetyl- d -glucosamine of LCOs is substituted at C₆ with 2- O -methyl- l -fucose which can be acetylated or sulphated. We identified a flavonoid-inducible locus on the symbiotic plasmid pNGR234 a that contains a new nodulation gene, noeE which is required for the sulphation of NGR234 Nod factors (NodNGR). noeE was identified by conjugation into the closely related Rhizobium fredii strain USDA257, which produces fucosylated but non-sulphated Nod factors (NodUSDA). R. fredii transconjugants producing sulphated LCOs acquire the capacity to nodulate Calopogonium caeruleum . Furthermore, mutation of noeE (NGRΔ noeE  ) abolishes the production of sulphated LCOs and prevents nodulation of Pachyrhizus tuberosus . The sulphotransferase activity linked to NoeE is specific for fucose. In contrast, the sulphotransferase NodH of Rhizobium meliloti seems to be less specific than NoeE, because its introduction into NGRΔ noeE leads to the production of a mixture of LCOs that are sulphated on C₆ of the reducing terminus and sulphated on the 2- O -methylfucose residue. Together, these findings show that noeE is a host-specificity gene which probably encodes a fucose-specific sulphotransferase.     

Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes.

Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far.