Genetic relationship of Sinorhizobium meliloti and Sinorhizobium medicae strains isolated from Caucasian region

Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species of the genus Sinorhizobium showing a similar host range, nodulating leguminous species of the genera Medicago, Melilotus and Trigonella, but their phylogenic relationship has not been elucidated yet. In this paper we report the application of three different molecular markers, (i) RFLP of nodD genes, (ii) 16S-23S rDNA intergenic gene spacer fingerprinting and (iii) amplification fragment length polymorphism to S. meliloti and S. medicae strains isolated from the Caucasian area, which is the region of origin of the host plant Medicago. The analysis of data could suggest the origin of S. medicae strains from an ancestral S. meliloti population.     

The stachydrine catabolism region in Sinorhizobium meliloti encodes a multi-enzyme complex similar to the xenobiotic degrading systems in other bacteria

Stachydrine (proline betaine) can be used by Sinorhizobium meliloti as a source of carbon and nitrogen. Catabolism depends on an initial N-demethylation, after which the resultant N-methyl proline enters general metabolism. Deletion and insertion mutagenesis demonstrated that the information necessary for catabolism is carried on the symbiotic plasmid (pSym) distal to nodD2 and the nod-nif cluster. Sequencing of an 8.5kb fragment spanning this region revealed four open reading frames with functional homology to known proteins, including a putative monooxygenase and a putative NADPH-FMN-reductase, which were shown by insertional and frame-shift mutagenesis to be necessary for stachydrine catabolism. Other open reading frames, encoding a putative flavoprotein and a repressor, were judged not to be required for stachydrine catabolism, since they were not included in a fragment capable of complementing a deletion of the entire stc region. Sequence and mutagenesis data suggest that stachydrine is demethylated by an iron-sulfur monooxygenase of the Rieske type with a requirement for a specific reductase. The stc catabolic cluster, therefore, resembles xenobiotic degradation in other bacteria and recalls rhizopine catabolism in S. meliloti. Stachydrine appears to have multiple roles in osmoprotection, nutrition and nodulation. Genes involved in stachydrine catabolism are also necessary for carnitine degradation; thus, they could be important in the catabolism of a variety of root exudates and mediate other relationships.     

Genetic analysis of a region of the Rhizobium meliloti pSym plasmid specifying catabolism of trigonelline, a secondary metabolite present in legumes.

Genes controlling the catabolism of trigonelline, a secondary metabolite that is often present in legumes, are located on the pSym megaplasmid of Rhizobium meliloti. To investigate the role of bacterial trigonelline catabolism in the Rhizobium-legume symbiosis, we identified and characterized the R. meliloti RCR2011 genetic loci (trc) controlling trigonelline catabolism. Tn5-B20 mutagenesis showed that the trc region is a continuous DNA segment of 9 kb located 4 kb downstream of the nifAB and fdxN genes. Trc mutants fell into two classes according to their phenotype and location: (i) mutants carrying Tn5-B20 insertions in the right-hand part of the trc region were incapable of growing on trigonelline as the sole carbon and/or nitrogen source, and (ii) insertions in the left-hand part of the trc region resulted in delayed growth on trigonelline as the sole carbon and/or nitrogen source. No significant defect in nodule formation or nitrogen fixation was detected for mutants of either class. Screening of a set of R. meliloti strains from various geographical origins showed that all of these strains are able to catabolize trigonelline and show sequence homology between their megaplasmids and a trc probe.     

苜蓿中华根瘤菌042BMnoeA基因的克隆、敲除与功能的初步分析

通过PCR扩增获得了 0 4 2BM的noeA基因。该基因与苜蓿中华根瘤菌 (Sinorhizobiummeliloti) 10 2 1noeA的同源性为 99% ,而其NoeA与 10 2 1NoeA的相似性为 97%。还发现其NoeA与中慢生根瘤菌 (Mesorhizobiumsp .)BNC1可能的SAM_依赖性的甲基转移酶相似性为 32 % ,而其 30 3～ 36 2氨基酸区域与大肠杆菌 (Escherichiacoli)的核糖体 5 0S亚基的L11蛋白甲基转移酶 (PrmA)的 16 0～ 2 2 0氨基酸区域的相似性达到 4 1%。通过插入卡那盒 ,敲除noeA ,获得突变株 0 4 2BMA_Km。与苜蓿中华根瘤菌 0 4 2BM相比 ,敲除noeA的突变株在普通紫花、保定、宁夏、百发和傲汉苜蓿品种上的结瘤数、根瘤鲜重和植株地上部分的干重都有不同程度的增加 ,而在秘鲁苜蓿品种上的结瘤数和植株地上部分的干重明显下降 ,在皇后和美国杂花苜蓿品种上则没有明显的变化。     

Genetic and biochemical characterization of a pathway for the degradation of 2-aminoethylphosphonate in Sinorhizobium meliloti 1021

A variety of microorganisms have the ability to use phosphonic acids as sole sources of phosphorus. Here, a novel pathway for degradation of 2-aminoethylphosphonate in the bacterium Sinorhizobium meliloti 1021 is proposed based on the analysis of the genome sequence. Gene deletion experiments confirmed the involvement of the locus containing phnW, phnA, and phnY genes in the conversion of 2-aminoethylphosphonate to inorganic phosphate. Biochemical studies of the recombinant PhnY and PhnA proteins verified their roles as phosphonoacetaldehyde dehydrogenase and phosphonoacetate hydrolase, respectively. This pathway is likely not limited to S. meliloti as suggested by the presence of homologous gene clusters in other bacterial genomes.     

Genetic organization and conjugal plasmid DNA transfer of pHP69, a plasmid from a Korean isolate of Helicobacter pylori

We isolated pHP69, a 9,153 bp plasmid from Helicobacter pylori with a 33.98% (G+C) content. We identified 11 open reading frames (ORFs), including replication initiation protein A (repA), fic (cAMP-induced filamentation protein), mccC, mccB, mobA, mobD, mobB, and mobC, as well as four 22 bp tandem repeat sequences. The nucleic acid and predicted amino acid sequences of these ORFs exhibited significant homology to those of other H. pylori plasmids. pHP69 repA encodes a replication initiation protein and its amino acid sequence is similar to those of replicase proteins from theta-type plasmids. pHP69 contains two types of repeat sequences (R1 and R2), a MOBHEN family mobilization region comprising mobC, mobA, mobB, and mobD, and genes encoding microcin B and C. Among the 36 H. pylori strains containing plasmids, mobA or mccBC are present in 12 or 6, respectively and 3 contain both genes. To examine intrinsic capability of H. pylori for conjugative plasmid transfer, a shuttle vector pBHP69KH containing pHP69 and replication origin of pBR322 was constructed. It was shown that this vector could stably replicate and be mobilized among clinical H. pylori strains and demonstrated to gene transfer by natural plasmid.     

Characterization and immobilization of a novel SGNH hydrolase (Est24) from Sinorhizobium meliloti

A novel oligomeric SGNH hydrolase (Est24) from Sinorhizobium meliloti was identified, actively expressed in Escherichia coli, characterized, and immobilized for industrial application. Sequence analysis of Est24 revealed a putative catalytic triad (Ser¹³-Asp¹⁶³-His¹⁶⁹), with moderate homology to other SGNH hydrolases. Est24 was more active toward short-chain esters, such as p-nitrophenyl acetate, butyrate, and valerate, while the S13A mutant completely lost its activity. Moreover, the activity of Est24 toward α- and β-naphthyl acetate, and enantioselectivity on (R)- and (S)-methyl-3-hydroxy-2-methylpropionate were tested. Est24 exhibited optimum activity at mesophilic temperature ranges (45–55 °C), and slightly alkaline pH (8.0). Structural and mutagenesis studies revealed critical residues involved in the formation of a catalytic triad and substrate-binding pocket. Cross-linked enzyme aggregates (CLEAs) of Est24 with and without amyloid fibrils were prepared, and amyloid fibril-linked Est24 with amyloid fibrils retained 83 % of its initial activity after 1 h of incubation at 60 °C. The high thermal stability of immobilized Est24 highlights its potential in the pharmaceutical and chemical industries.     

Genetic diversity of a natural population of Sinorhizobium meliloti, detected during analysis of a cryptic plasmid and ISRm2011-2 fingerprints]

Fifty-six natural strains of alfalfa nodule bacteria were isolated from samples of the soil under wild legume and alfalfa in two different field sites of Irkutsk oblast. Based on the results of analysis of plasmid profile, 11 different types of strains were detected, and 43 types were identified based on the results of hybridization with the insertion sequence element ISRm2011-2. Significant differences were found in the plasmid profile and IS fingerprints between strains isolated from the soil under alfalfa and the soil under legume. In contrast, strains growing at some distance from each other differed only in the IS fingerprints. From a comparison of results obtained in the assessment of plasmid profile and in analysis of IS fingerprints with results of RFLP analysis in strains, the conclusion about the transference of cryptic plasmids between strains and genetic rearrangements in strains of this population was drawn.     

Cloning and characterization of a second acid phosphatase from Sinorhizobium meliloti strain 104A14

Sinorhizobium meliloti has two nonspecific periplasmic acid phosphatases. The NapD enzyme has been previously described, and a second acid phosphatase, NapE, is described in this report. NapE was partially purified from an S. meliloti napD mutant and characterized with respect to molecular mass and substrate range. As predicted from SDS-PAGE analysis, the subunit molecular mass of NapE is approximately 35.8 kDa and gel filtration experiments estimated the native molecular mass to be approximately 70 kDa, indicating that the active enzyme is a homodimer. NapE demonstrated significant activity with p-nitrophenyl phosphate, phenyl phosphate, and alpha-naphthyl-phosphate. The pH optimum was between 4.5 and 5.0. The gene encoding NapE was also sequenced and the inferred amino acid sequence from the predicted ORF was found to be 60% identical and 75% similar to that encoded by napD. An S. meliloti napE mutant was constructed and assessed for symbiotic competence. This mutant did not differ from the wild-type parent strain in nodulation and symbiotic efficiency.     

Design,structural and functional characterization of a Temporin-1b analog active against Gram-negative bacteria

Background

Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs.

Methods

Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB_G6A sequence, which was further optimized by increasing the peptide positive charge (TB_KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR.

Results

TB_KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix.

Conclusion

Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, a broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides.

General significance

The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipathic, since this feature seems to help the toroidal pore formation process.     

Molecular cloning, purification, and biochemical characterization of hydantoin racemase from the legume symbiont Sinorhizobium meliloti CECT 4114

Hydantoin racemase from Sinorhizobium meliloti was functionally expressed in Escherichia coli. The native form of the enzyme was a homotetramer with a molecular mass of 100 kDa. The optimum temperature and pH for the enzyme were 40 degrees C and 8.5, respectively. The enzyme showed a slight preference for hydantoins with short rather than long aliphatic side chains or those with aromatic rings. Substrates, which showed no detectable activity toward the enzyme, were found to exhibit competitive inhibition.     

The Symbiosis Interactome: a computational approach reveals novel components,functional interactions and modules in Sinorhizobium meliloti

Background  

Rhizobium-Legume symbiosis is an attractive biological process that has been studied for decades because of its importance in agriculture. However, this system has undergone extensive study and although many of the major factors underpinning the process have been discovered using traditional methods, much remains to be discovered.     

Genetic basis of enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprint pattern in Sinorhizobium meliloti and identification of S. meliloti employing PCR primers derived from an ERIC-PCR fragment

The enterobacterial repetitive intergenic consensus (ERIC)-PCR method was employed to generate genomic amplification products of Sinorhizobium meliloti strain 2011. Eleven distinctive PCR fragments obtained in PCR reactions by using the ERIC2 primer were cloned and their partial or complete nucleotide sequences established. DNA sequences that extended past the ERIC2 primer region were not conserved among the 11 PCR fragments and showed no sequence similarity to the enterobacterial ERIC consensus sequence. Thus, repetitive ERIC or ERIC-like sequences seem not to be an integral part of the S. meliloti genome. An amplification product of S. meliloti 2011 was identified which was present in S. meliloti strains but absent in other rhizobial species. Based on the nucleotide sequence information, a pair of PCR primers was designed and used for PCR amplification of sequences of S. meliloti laboratory strains 2011, L5–30, AK631 and 102F34. Nucleotide sequence analysis of the amplification products revealed a 100% DNA sequence conservation. Database searches showed that the DNA fragment putatively encodes the C-terminal part of a protein displaying similarity to 2-hydroxyacid dehydrogenases of various organisms. The newly designed PCR primers should be useful for the rapid identification of S. meliloti isolates. Received: 17 February 1999 / Accepted: 9 April 1999