Structural and functional analysis of the riboflavin synthesis genes encoding GTP cyclohydrolase II (ribA), DHBP synthase (ribBA), riboflavin synthase (ribC), and riboflavin deaminase/reductase (ribD) from Helicobacter pylori strain P1

The functions of the riboflavin synthesis gene homologues ribA, ribBA, ribC, and ribD from Helicobacter pylori strain P1 were confirmed by complementation of defined Escherichia coli mutant strains. The H. pylori ribBA gene, which is similar to bifunctional ribBA genes of Gram-positive bacteria, fully complemented the ribB mutation and partially restored growth in a ribC mutant. However, ribBA did not complement the ribA mutation in E. coli, thus explaining the presence of the additional separate copy of the ribA gene in the H. pylori chromosome. In E. coli exclusively ribA conferred hemolytic activity and gave rise to production of molecules with fluorescence characteristics similar to flavins, as observed earlier. The E. coli hemolysin ClyA was not involved in causing the hemolytic phenotype. No riboflavin synthesis genes on plasmids conferred iron uptake functions to a siderophore-deficient mutant of E. coli. Marker exchange mutagenesis of the genes in H. pylori was not successful indicating that riboflavin synthesis is essential for basic metabolic functions of the gastric pathogen.     

Molecular evidence of genetic modification of Sinorhizobium meliloti: enhanced PCB bioremediation

The genome of the nitrogen-fixing soil bacterium Sinorhizobium meliloti does not possess genes for bioremediation of aromatic pollutants. It has the well-known ability to interact specifically with the leguminous alfalfa plant, Medicago sativa. Our previous work has shown enhanced degradation of the nitroaromatic compound 2,4-dinitrotoluene (DNT) when a plasmid containing degradative genes was introduced in it. In this study we report molecular evidence of the transfer of a polychlorinated biphenyl (PCB)-biodegradative plasmid pE43 to S. meliloti strain USDA 1936. Several standard analytical tests and plant growth chamber studies were conducted to test the ability of S. meliloti to degrade 2',3,4-PCB congener. Alfalfa plant alone was able to degrade 30% of PCBs compared with control. No enhanced dechlorination was noted when alfalfa plant was grown with wild-type S. meliloti, and when alfalfa plant was grown with the S. meliloti electrotransformants (genetically modified) dechlorination of PCBs was more than twice that when alfalfa plant was grown with wild-type S. meliloti. When alfalfa plant was grown with uncharacterized mixed culture (containing nodule formers), almost equally significant PCB degradation was observed. The significance of this work is that the naturally occurring nitrogen-fixing soil bacterium S. meliloti (genetically modified) has the ability to enhance fertility of soil in association with the leguminous alfalfa plant while simultaneously enhancing bioremediation of PCB-contaminated soils. Enhanced bioremediation of PCB and robust alfalfa plant growth was also noted when uncharacterized mixed cultures containing alfalfa plant nodule formers were used.     

Functional analysis of Sinorhizobium meliloti genes involved in biotin synthesis and transport

External biotin greatly stimulates bacterial growth and alfalfa root colonization by Sinorhizobium meliloti strain 1021. Several genes involved in responses to plant-derived biotin have been identified in this bacterium, but no genes required for biotin transport are known, and not all loci required for biotin synthesis have been assigned. Searches of the S. meliloti genome database in combination with complementation tests of Escherichia coli biotin auxotrophs indicate that biotin synthesis probably is limited in S. meliloti 1021 by the poor functioning or complete absence of several key genes. Although several open reading frames with significant similarities to genes required for synthesis of biotin in gram-positive and gram-negative bacteria were found, only bioB, bioF, and bioH were demonstrably functional in complementation tests with known E. coli mutants. No sequence or complementation evidence was found for bioA, bioC, bioD, or bioZ. In contrast to other microorganisms, the S. meliloti bioB and bioF genes are not localized in a biotin synthesis operon, but bioB is cotranscribed with two genes coding for ABC transporter-like proteins, designated here bioM and bioN. Mutations in bioM and bioN eliminated growth on alfalfa roots and reduced bacterial capacity to maintain normal intracellular levels of biotin. Taken together, these data suggest that S. meliloti normally grows on exogenous biotin using bioM and bioN to conserve biotin assimilated from external sources.     

Early symbiotic responses induced by Sinorhizobium meliloti iIvC mutants in alfalfa

A mutation in the ilvC gene of Sinorhizobium meliloti 1021 determines a symbiotically defective phenotype. ilvC mutants obtained from different S. meliloti wild-type strains are able to induce root hair deformation on alfalfa roots and show variable activation of the common nodulation genes nodABC. All of these mutants are noninfective. The presence of extra copies of nodD3-syrM in an IlvC- background does not promote nod expression but allows the detection of low levels of Nod factor production. The sulphation of the Nod factor metabolites, however, is not affected. Furthermore, IlvC- strains induce a specific pattern of starch accumulation on alfalfa roots as well as of early nodulin expression. Hence, the pleiotropic action of the ilvC gene in S. meliloti may reveal novel complexities involved in the symbiotic interaction.     

Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.     

Increase in alfalfa nodulation, nitrogen fixation, and plant growth by specific DNA amplification in Sinorhizobium meliloti.

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

Sugar-binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants by Rhizobium leguminosarum biovar viciae

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo(-) R. leguminosarum bv viciae strain that produced S. meliloti Nod factor.     

Identification of Sinorhizobium meliloti early symbiotic genes by use of a positive functional screen

The soil bacterium Sinorhizobium meliloti establishes nitrogen-fixing symbiosis with its leguminous host plant, alfalfa, following a series of continuous signal exchanges. The complexity of the changes of alfalfa root structures during symbiosis and the amount of S. meliloti genes with unknown functions raised the possibility that more S. meliloti genes may be required for early stages of the symbiosis. A positive functional screen of the entire S. meliloti genome for symbiotic genes was carried out using a modified in vivo expression technology. A group of genes and putative genes were found to be expressed in early stages of the symbiosis, and 23 of them were alfalfa root exudate inducible. These 23 genes were further separated into two groups based on their responses to apigenin, a known nodulation (nod) gene inducer. The group of six genes not inducible by apigenin included the lsrA gene, which is essential for the symbiosis, and the dgkA gene, which is involved in the synthesis of cyclic beta-1,2-glucan required for the S. meliloti-alfalfa symbiosis. In the group of 17 apigenin-inducible genes, most have not been previously characterized in S. meliloti, and none of them belongs to the nod gene family. The identification of this large group of alfalfa root exudate-inducible S. meliloti genes suggests that the interactions in the early stages of the S. meliloti and alfalfa symbiosis could be complex and that further characterization of these genes will lead to a better understanding of the symbiosis.     

Phosphorus-free membrane lipids of Sinorhizobium meliloti are not required for the symbiosis with alfalfa but contribute to increased cell yields under phosphorus-limiting conditions of growth

The microsymbiont of alfalfa, Sinorhizobium meliloti, possesses phosphatidylglycerol, cardiolipin, phosphatidylethanolamine, and phosphatidylcholine as major membrane phospholipids, when grown in the presence of sufficient accessible phosphorus sources. Under phosphate-limiting conditions of growth, S. meliloti replaces its phospholipids by membrane lipids that do not contain any phosphorus in their molecular structure and, in S. meliloti, these phosphorus-free membrane lipids are sulphoquinovosyl diacylglycerols (SL), ornithine-containing lipids (OL), and diacylglyceryl-N,N,N-trimethylhomoserines (DGTS). In earlier work, we demonstrated that neither SL nor OL are required for establishing a nitrogen-fixing root nodule symbiosis with alfalfa. We now report the identification of the two structural genes btaA and btaB from S. meliloti required for DGTS biosynthesis. When the sinorhizobial btaA and btaB genes are expressed in Escherichia coli, they cause the formation of DGTS in this latter organism. A btaA-deficient mutant of S. meliloti is unable to form DGTS but can form nitrogen-fixing root nodules on alfalfa, demonstrating that sinorhizobial DGTS is not required for establishing a successful symbiosis with the host plant. Even a triple mutant of S. meliloti, unable to form any of the phosphorus-free membrane lipids SL, OL, or DGTS is equally competitive for nodule occupancy as the wild type. Only under growth-limiting concentrations of phosphate in culture media did mutants that could form neither OL nor DGTS grow to lesser cell densities.     

一株能在大豆上结瘤的苜蓿中华根瘤菌

苜蓿中华根瘤菌(Sinorhizobium meliloti)XJ96077分离自新疆的苜蓿根瘤中,其原宿主为紫花苜蓿(Medicago sativa)。交叉结瘤试验发现,它既可在苜蓿上又能在大豆上结瘤固氮。DNA(G C)mol％分析表明,XJ96077的DNA(G C)mol％为61．9％,与已报道的根瘤菌属的DNA(G C)mol％范围(59％-64％)相符。DNA同源性分析表明,XJ96077与苜蓿中华根瘤菌USDA1002^T和042BM的同源性分别达到93％和80％,说明XJ96077归属于苜蓿中华根瘤菌。应用绿色荧光蛋白基因标记XJ96077,得到重组菌株XJ96077(G)。将其接种普通紫花苜蓿,通过激光共聚焦荧光显微镜可以检测到标记基因的表达。接种北引1号大豆上,同样可以清楚地观察到标记基因在根瘤中的表达,从而确证了XJ96077能同时在苜蓿和大豆上结瘤。通过不同品种大豆的结瘤试验,发现XJ96077对大豆品种的结瘤能力不同。     

苜蓿根瘤菌17560细胞膜膜脂组成分析及DGTS合成基因克隆与表达

【目的】分析一株分离自黑龙江省的苜蓿根瘤菌在低磷胁迫及正常磷含量条件下细胞膜脂的组成,并从该菌中克隆和鉴定细胞膜无磷脂二酰基甘油三甲基高丝氨酸(DGTS)合成基因。【方法】分别在不同磷含量的Sherwood基本培养基中进行根瘤菌培养,采用Bligh-Dyer方法提取细胞膜脂,以文献报道Sinorhizobium meliloti(苜蓿中华根瘤菌)菌株1021的脂类图谱和磷脂PE、PG、PC标准品作为参照,利用薄层层析方法分析不同磷含量条件下培养菌株的细胞膜脂组成。根据GenBank中已发表的DGTS合成基因btaA和btaB序列设计引物,以产DGTS菌株基因组DNA为模板,扩增btaA和btaB同源基因,并在E.coil BL21(DE3)表达。同时检测表达菌株是否合成细胞膜无磷脂DGTS以验证基因功能。对菌株17560进行16S rRNA基因序列分析。【结果】分离自黑龙江省的苜蓿根瘤菌17560与Sinorhizobium meliloti的16S rRNA基因序列相似性高达99.8%,但其细胞膜脂组成明显不同于参比菌株Sinorhizobium meliloti 1021的膜脂组成。在低磷胁迫条件下,该菌株的细胞膜脂主要由OL和DGTS等无磷脂组成,但OL的组成明显不同,该菌株含有3种不同类型的鸟氨酸脂(OLs),而参比菌株Sinorhizobium meliloti 1021只含有一种类型的鸟氨酸脂(OL)。在正常磷含量条件下,该菌株的细胞膜脂主要由PE和一种未知的含氨基磷脂组成,PG与PC的含量均较少,而参比菌株Sinorhizobium meliloti 1021的细胞膜脂主要由PE、PG与PC组成。通过PCR扩增从产DGTS菌株17560中获得1 913 bpDNA片段,经序列分析发现其中有两个ORF与菌株Sinorhizobium meliloti 1021的btaA和btaB基因序列相似性均为99%。将该DNA片段克隆于pET-30a(+)得到重组质粒pLH01,转化宿主菌获得表达菌株E.coli BL21(DE3).pLH01,经IPTG诱导后产生相对分子量约为45 kD和25 kD的蛋白。薄层层析验证重组菌细胞膜脂组成,结果表明,表达菌株E.coliBL21(DE3).pLH01可以在IPTG诱导后合成无磷脂DGTS,而转入空载体pET-30a(+)的阴性对照菌株E.coli BL21(DE3).pET-30a(+)则不能合成。【结论】系统发育地位相同的苜蓿根瘤菌株的细胞膜脂组成明显不同;苜蓿根瘤菌的细胞膜组成随培养基中的磷含量不同而变化,低磷胁迫条件下其细胞膜脂主要由OL和DGTS等无磷脂组成;在Sinorhizobium膜脂中首次发现一种未知的氨基磷脂及3种不同类型的鸟氨酸脂(OLs);从菌株17560中克隆获得2个DGTS合成基因btaA和btaB,在大肠杆菌中成功表达,并证实了所表达基因的功能。     

Two RpoH homologs responsible for the expression of heat shock protein genes in Sinorhizobium meliloti

We identified two rpoH-related genes encoding sigma32-like proteins from Sinorhizobium meliloti, a nitrogen-fixing root-nodule symbiont of alfalfa. The genes, rpoH1 and rpoH2, are functionally similar to rpoH of Escherichia coli because they partially complemented an E. coli rpoH null mutant. We obtained evidence indicating that these genes are involved in the heat shock response in S. meliloti. Following an increase in temperature, synthesis of several putative heat shock proteins (Hsps) was induced in cultures of wild-type cells: the most prominent were 66- and 60-kDa proteins, both of which are suggested to represent GroEL species. The other Hsps could divided into two groups based on differences in synthesis kinetics: synthesis of the first group peaked 5-10 min, and expression of the other group 30 min, after temperature upshift. In the rpoH1 mutant, inducible synthesis of the former group was markedly reduced, whereas that of the latter group was not affected. Synthesis of both the 66- and 60-kDa proteins was partially reduced. While no appreciable effect was observed in the rpoH2 single mutant, the rpoH2 mutation had a synergistic effect on the 60-kDa protein in the rpoH1- background. The results indicate that two distinct mechanisms are involved in the heat shock response of S. meliloti: one requires the rpoH1 function, while rpoH2 can substitute in part for the rpoH1 function. Moreover, the rpoH1 mutant and rpoH1 rpoH2 double mutant exhibited Nod+ Fix- and Nod- phenotypes, respectively, on alfalfa.     

The biological activity of the Sinorhizobium meliloti glucan

The study of the effect of the periplasmic glucan isolated from the root-nodule bacterium S. meliloti CXM1-188 on the symbiosis of another strain (441) of the same root-nodule bacterium with alfalfa plants showed that this effect depends on the treatment procedure. The pretreatment of alfalfa seedlings with the glucan followed by their bacterization with S. meliloti 441 insignificantly influenced the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity) but induced a statistically significant increase in the efficiency of symbiosis (expressed as the masses of the alfalfa overground parts and roots). At the same time, the pretreatment of S. meliloti 441 cells with the glucan brought about a considerable decrease in the nodulation parameters of symbiosis (the number of the root nodules and their nitrogen-fixing activity decreased by 2.5-11 and 7 times, respectively). These data suggest that the stimulating effect of rhizobia on host plants may be due not only to symbiotrophic nitrogen fixation but also to other factors. Depending on the experimental conditions, the treatment of alfalfa plants with the glucan and their bacterization with rhizobial cells enhanced the activity of peroxidase in the alfalfa roots and leaves by 10-39 and 12-27%, respectively.     

The product of the Rhizobium meliloti ilvC gene is required for isoleucine and valine synthesis and nodulation of alfalfa.

Tn5-induced mutants of Rhizobium meliloti that require the amino acids isoleucine and valine for growth on minimal medium were studied. In one mutant, 1028, the defect is associated with an inability to induce nodules on alfalfa. The Tn5 mutation in 1028 is located in a chromosomal 5.5-kb EcoRI fragment. Complementation analysis with cloned DNA indicated that 2.0 kb of DNA from the 5.5-kb EcoRI fragment restored the wild-type phenotype in the Ilv- Nod- mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to those for Escherichia coli IlvC and Saccharomyces cerevisiae Ilv5. Genes ilvC and ilv5 code for the enzyme acetohydroxy acid isomeroreductase (isomeroreductase), the second enzyme in the parallel pathways for the biosynthesis of isoleucine and valine. Enzymatic assays confirmed that strain 1028 was a mutant defective in isomeroreductase activity. In addition, it was shown that the ilvC genes of Rhizobium meliloti and E. coli are functionally equivalent. We demonstrated that in ilvC mutant 1028 the common nodulation genes nodABC are not activated by the inducer luteolin. E. coli ilvC complemented both defective properties (Ilv- and Nod-) found in mutant 1028. These findings demonstrate that R. meliloti requires an active isomeroreductase enzyme for successful nodulation of alfalfa.     

Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species.

Analysis of the DNA regions upstream of the phosphoenolpyruvate carboxykinase gene (pckA) in Rhizobium meliloti and Rhizobium sp. strain NGR234 identified an open reading frame which was highly homologous to the Agrobacterium tumefaciens chromosomal virulence gene product ChvI. A second gene product, 500 bp downstream of the chvI-like gene in R. meliloti, was homologous to the A. tumefaciens ChvG protein. The homology between the R. meliloti and A. tumefaciens genes was confirmed, because the R. meliloti chvI and chvG genes complemented A. tumefaciens chvI and chvG mutants for growth on complex media. We were unable to construct chvI or chvG insertion mutants of R. meliloti, whereas mutants carrying insertions outside of these genes were readily obtained. A 108-bp repeat element characterized by two large palindromes was identified in the chvI and chvG intergenic regions of both Rhizobium species. This element was duplicated in Rhizobium sp. strain NGR234. Another structurally similar element with a size of 109 bp was present in R. meliloti but not in Rhizobium sp. strain NGR234. These elements were named rhizobium-specific intergenic mosaic elements (RIMEs), because their distribution seems to be limited to members of the family Rhizobiaceae. A homology search in GenBank detected six more copies of the first element (RIME1), all in Rhizobium species, and three extra copies of the second element (RIME2), only in R. meliloti. Southern blot analysis with a probe specific to RIME1 showed the presence of several copies of the element in the genome of R. meliloti, Rhizobium sp. strain NGR234, Rhizobium leguminosarum, and Agrobacterium rhizogenes, but none was present in A. tumefaciens and Bradyrhizobium japonicum.     

Characterization of bdhA, encoding the enzyme D-3-hydroxybutyrate dehydrogenase, from Sinorhizobium sp. strain NGR234

A genomic library of Sinorhizobium sp. strain NGR234 was introduced into Escherichia coli LS5218, a strain with a constitutively active pathway for acetoacetate degradation, and clones that confer the ability to utilize D-3-hydroxybutyrate as a sole carbon source were isolated. Subcloning experiments identified a 2.3 kb EcoRI fragment that retained complementing ability, and an ORF that appeared orthologous with known bdhA genes was located within this fragment. The deduced NGR234 BdhA amino acid sequence revealed 91% identity to the Sinorhizobium meliloti BdhA. Site-directed insertion mutagenesis was performed by introduction of a OmegaSmSp cassette at a unique EcoRV site within the bdhA coding region. A NGR234 bdhA mutant, NGRPA2, was generated by homogenotization, utilizing the sacB gene-based lethal selection procedure. This mutant was devoid of D-3-hydroxybutyrate dehydrogenase activity, and was unable to grow on D-3-hydroxybutyrate as sole carbon source. NGRPA2 exhibited symbiotic defects on Leucaena but not on Vigna, Macroptilium or Tephrosia host plants. Furthermore, the D-3-hydroxybutyrate utilization phenotype of NGRPA2 was suppressed by presence of plasmid-encoded multiple copies of the S. meliloti acsA2 gene. The glpK-bdhA-xdhA gene organization and the bdhA-xdhA operon arrangement observed in S. meliloti are also conserved in NGR234.