Broad host range and promoter selection vectors for bacteria that interact with plants.

A plasmid vector, pGV910, and a derived cosmid, pRG930, have been constructed. Both contain the ColE1 and pVS1 origins of replication and are stably maintained in Escherichia coli, Agrobacterium tumefaciens, and Azorhizobium caulinodans ORS571. They are compatible with commonly used IncP cloning vectors, although pVS1 was classified as an IncP plasmid, unable to replicate in E. coli (Y. Itoh, J.M. Watson, D. Haas, and T. Leisinger, Plasmid 11:206-220, 1984). Promoter selection vectors were derived from both of these plasmids by using a promoterless beta-glucuronidase and/or beta-galactosidase gene. These vectors facilitate the study of gene expression in bacteria under particular environmental conditions. This is illustrated by the expression of the gusA gene under the control of a nod promoter in A. caulinodans nodulating stem-located infection sites on Sesbania rostrata.     

Nod factor perception during infection thread growth fine-tunes nodulation

Bacterial nodulation factors (NFs) are essential signaling molecules for the initiation of a nitrogen-fixing symbiosis in legumes. NFs are perceived by the plant and trigger both local and distant responses, such as curling of root hairs and cortical cell divisions. In addition to their requirement at the start, NFs are produced by bacteria that reside within infection threads. To analyze the role of NFs at later infection stages, several phases of nodulation were studied by detailed light and electron microscopy after coinoculation of adventitious root primordia of Sesbania rostrata with a mixture of Azorhizobium caulinodans mutants ORS571-V44 and ORS571-X15. These mutants are deficient in NF production or surface polysaccharide synthesis, respectively, but they can complement each other, resulting in functional nodules occupied by ORS571-V44. The lack of NFs within the infection threads was confirmed by the absence of expression of an early NF-induced marker, leghemoglobin 6 of S. rostrata. NF production within the infection threads is shown to be necessary for proper infection thread growth and for synchronization of nodule formation with bacterial invasion. However, local production of NFs by bacteria that are taken up by the plant cells at the stage of bacteroid formation is not required for correct symbiosome development.     

Gibberellins are involved in nodulation of Sesbania rostrata

Upon submergence, Azorhizobium caulinodans infects the semiaquatic legume Sesbania rostrata via the intercellular crack entry process, resulting in lateral root-based nodules. A gene encoding a gibberellin (GA) 20-oxidase, SrGA20ox1, involved in GA biosynthesis, was transiently up-regulated during lateral root base nodulation. Two SrGA20ox1 expression patterns were identified, one related to intercellular infection and a second observed in nodule meristem descendants. The infection-related expression pattern depended on bacterially produced nodulation (Nod) factors. Pharmacological studies demonstrated that GAs were involved in infection pocket and infection thread formation, two Nod factor-dependent events that initiate lateral root base nodulation, and that they were also needed for nodule primordium development. Moreover, GAs inhibited the root hair curling process. These results show that GAs are Nod factor downstream signals for nodulation in hydroponic growth.     

Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from Gram-positive organisms

A 2.6 kb plasmid, named pBBR1, was isolated from Bordetella bronchiseptica S87. After insertion of an antibiotic resistance marker, this plasmid could be transferred into Escherichia coli, Bordetella pertussis, B. bronchiseptica, Vibrio cholerae, Rhizobium meliloti, and Pseudomonas putida by transformation or conjugation. Conjugation was possible only when the IncP group transfer functions were provided in trans. As shown by incompatibility testing, pBBR1 does not belong to the broad-host-range IncP, IncQ or IncW groups. DNA sequence analysis revealed two open reading frames: one was called Rep, involved in replication of the plasmid, and the other, called Mob, was involved in mobilization. Both the amino-terminal region of Mob and its promoter region show sequence similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. In spite of these sequence similarities, pBBR1 does not replicate via the rolling-circle mechanism commonly used by small Gram-positive plasmids. We therefore speculate that pBBR1 may combine a mobilization mechanism of Gram-positive organisms with a replication mechanism of Gram-negative organisms. Determination of the plasmid copy number in E. coli and B. pertussis indicated that pBBR1 has a rather high copy number, which, in conjunction with its small size and broad host range, renders it particularly interesting for studies of broad-host-range replicons and for the development of new cloning vectors for a wide range of Gram-negative bacteria.     

Root nodulation of Sesbania rostrata.

The tropical legume Sesbania rostrata can be nodulated by Azorhizobium caulinodans on both its stem and its root system. Here we investigate in detail the process of root nodulation and show that nodules develop exclusively at the base of secondary roots. Intercellular infection leads to the formation of infection pockets, which then give rise to infection threads. Concomitantly with infection, cortical cells of the secondary roots dedifferentiate, forming a meristem which has an "open-basket" configuration and which surrounds the initial infection site. Bacteria are released from the tips of infection threads into plant cells via "infection droplets," each containing several bacteria. Initially, nodule differentiation is comparable to that of indeterminate nodules, with the youngest meristematic cells being located at the periphery and the nitrogen-fixing cells being located at the nodule center. Because of the peculiar form of the meristem, Sesbania root nodules develop uniformly around a central axis. Nitrogen fixation is detected as early as 3 days following inoculation, while the nodule meristem is still active. Two weeks after inoculation, meristematic activity ceases, and nodules then show the typical histology of determinate nodules. Thus, root nodule organogenesis in S. rostrata appears to be intermediate between indeterminate and determinate types.     

Structural characterization of extracellular polysaccharides of Azorhizobium caulinodans and importance for nodule initiation on Sesbania rostrata

During lateral root base nodulation, the microsymbiont Azorhizobium caulinodans enters its host plant, Sesbania rostrata, via the formation of outer cortical infection pockets, a process that is characterized by a massive production of H(2)O(2). Infection threads guide bacteria from infection pockets towards nodule primordia. Previously, two mutants were constructed that produce lipopolysaccharides (LPSs) similar to one another but different from the wild-type LPS, and that are affected in extracellular polysaccharide (EPS) production. Mutant ORS571-X15 was blocked at the infection pocket stage and unable to produce EPS. The other mutant, ORS571-oac2, was impaired in the release from infection threads and was surrounded by a thin layer of EPS in comparison to the wild-type strain that produced massive amounts of EPS. Structural characterization revealed that EPS purified from cultured and nodule bacteria was a linear homopolysaccharide of alpha-1,3-linked 4,6-O-(1-carboxyethylidene)-D-galactosyl residues. In situ H(2)O(2) localization demonstrated that increased EPS production during early stages of invasion prevented the incorporation of H(2)O(2) inside the bacteria, suggesting a role for EPS in protecting the microsymbiont against H(2)O(2). In addition, ex planta assays confirmed a positive correlation between increased EPS production and enhanced protection against H(2)O(2).     

Rhizobial factors required for stem nodule maturation and maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 symbiosis

The molecular and physiological mechanisms behind the maturation and maintenance of N(2)-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N(2)-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N(2) fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C(4) metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.     

Interaction of a rhizobial DNA-binding protein with the promoter region of a plant leghemoglobin gene.

A nucleotide sequence was identified approximately 650 bp upstream of the Sesbania rostrata leghemoglobin gene Srglb3 start codon, which interacts specifically with a proteinaceous DNA-binding factor found in nodule extracts but not in extracts from leaves or roots. The binding site for this factor was delimited using footprinting techniques. The DNA-binding activity of this factor was found to be heat stable, dependent on divalent cations, and derived from the (infecting) Azorhizobium caulinodans bacteria or bacteroids (A. caulinodans bacterial binding factor 1, AcBBF1). A 9- to 10-kD protein was isolated from a free-living culture of A. caulinodans that co-purifies with the DNA-binding activity (A. caulinodans bacterial binding protein 1, AcBBP1) and interacts specifically with its target (S. rostrata bacterial binding site 1, SrBBS1). The amino acid sequence of the N-terminal 27 residues of AcBBP1 was determined and was found to share significant similarity (46% identity; 68% similarity) with a domain of the herpes simplex virus major DNA-binding protein infected cell protein 8 (ICP8). An insertion mutation in the SrBBS1 was found to result in a substantial reduction of the expression of a Srglb3-gus reporter gene fusion in nodules of transgenic Lotus corniculatus plants, suggesting a role for this element in Srglb3 promoter activity. Based on these results, we propose that (a) bacterial transacting factor(s) may play a role in infected cell-specific expression of the symbiotically induced plant lb genes.     

苜蓿中华根瘤菌042B nodD基因的克隆、序列分析及其表达

苜蓿中华根瘤菌０４２Ｂ是一株能在苜蓿和大豆上结瘤的菌株。将０４２Ｂ的ｎｏｄＳＤ基因克隆到时载体ｐＢＢＲ１ＭＣＳ－５,并在豌豆根瘤菌ＬＲＲ５０４５系统中进行功能分析,发现０４２Ｂ的ＮｏｄＤ蛋白能与大豆的类黄酮化合物ｇｅｎｉｓｔｅｉｎ结合,也怀苜蓿原类黄酮化合物ｌｕｔｅｏｌｉｎ反应。     

Distribution of plasmids in cyanobacteria of the LPP group

Abstract The distribution of plasmid DNA has been studied in 23 strains and variants of non-heterocystous filamentous cyanobacteria that are susceptible to infection by the LPP-1 cyanophage (archetype). 21 have identical plasmid profiles and contain 3 plasmids of M _rs 0.9, 10 and approx. 12 · 10⁶ respectively. In one strain, Plectonema FS180, the plasmids have been designated pMP1, pMP2, and pMP3, respectively. pMP1 shows sequence homology with pMP2 and pMP3 but not with DNA from an LPP-type cyanophage. Plectonema UTEX 598 lacks the small plasmid only, while Plectonema UTEX 1541 is distinct amongst all these strains with 3 plasmids of M _rs 3, 10, and > 30 · 10⁶, respectively. The findings support the view that the majority of these strains may be independent isolates of a single species.     

A new root-nodulating symbiont of the tropical legume Sesbania, Rhizobium sp SIN-1, is closely related to R. galegae, a species that nodulates temperate legumes

Abstract Rhizobium sp. SIN-1, isolated in India from root nodules on the tropical legume Sesbania aculeata , also induces nitrogen-fixing nodules on roots of S. macrocarpa, S. speciosa, S. procumbens, S. punicea, S. rostrata , and Vigna unguiculata . Unlike Azorhizobium caulinodans , SIN-1 does not induce stem nodules on S. rostrata . The nodules induced by SIN-1 develop exclusively at the bases of secondary roots. Electron microscopic studies of mature nodule sections revealed rhizobia within intercellular spaces, indicating a 'crack entry' mechanism of root infection. SIN-1 is a fast-growing, acid-producing, salt-tolerant Rhizobium that utilizes a wide variety of carbon sources. The nodulation ( nod ) genes of this strain are located on a 300-MDa symbiosis ( sym ) plasmid. Fatty acid profile and sequence comparison of a 260-bp conserved region of the 16S rRNA gene demonstrated that SIN-1 is phylogenetically closely related to R. galegae , a species that nodulates temperate legumes.     

Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1

The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica (R. Antoine and C. Locht, Mol. Microbiol. 6:1785-1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the mob gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and mob gene regulation. We show that the Mob protein is a relaxase, and we located the nic site position in vitro. After sequence alignment, the position of the nic site of pBBR1 corresponds with those of the nick sites of the Bacteroides mobilizable transposon Tn4555 and the streptococcal plasmid pMV158. The oriT of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism.     

Interactions of rhizobia with rice and wheat

Recently, evidence has been obtained that naturally occurring rhizobia, isolated from the nodules of non-legume Parasponia species and from some tropical legumes, are able to enter the roots of rice, wheat and maize at emerging lateral roots by crack entry. We have now investigated whether Azorhizobium caulinodans strain ORS571, which induces root and stem nodules on the tropical legume Sesbania rostrata as a result of crack entry invasion of emerging lateral roots, might also enter rice and wheat by a similar route. Following inoculation with ORS571 carrying a lacZ reporter gene, azorhizobia were observed microscopically within the cracks associated with emerging lateral roots of rice and wheat. A high proportion of inoculated rice and wheat plants had colonized lateral root cracks. The flavanone naringenin at 10 and 10 M stimulated significantly the colonization of lateral root cracks and also intercellular colonization of wheat roots. Naringenin does not appear to be acting as a carbon source and may act as a signal molecule for intercellular colonization of rice and wheat by ORS571 by a mechanism which is nod gene-independent, unlike nodule formation in Sesbania rostrata. The opportunity now arises to compare and to contrast the ability of Azorhizobium caulinodans with that of other rhizobia, such as Parasponia rhizobia, to intercellularly colonize the roots of non-legume crops.     

Transfer of recombinant plasmids containing the gene for DpnII DNA methylase into strains of Streptococcus pneumoniae that produce DpnI or DpnII restriction endonucleases.

Plasmid transfer via the transformation pathway of Streptococcus pneumoniae was weakly restricted by the DpnI or DpnII restriction endonuclease, either of which gave a reduction only to 0.4, compared with phage infection, which was restricted to 10(-5). The greater sensitivity of plasmid transfer compared with chromosomal transformation, which was not at all restricted, can be attributed to partially double-stranded intermediates formed from two complementary donor fragments. However, clustering of potential restriction sites in the plasmids increased the probability of escape from restriction. The recombinant plasmid pMP10 , in which the gene for the DpnII DNA methylase was cloned, can be transferred to strains that contain neither restriction enzyme or that contain DpnII as readily as can the vector pMP5 . Introduction of pMP10 raised the level of methylase by five times the level normally present in DpnII strains. Transfer of pMP10 to DpnI -containing strains was infrequent, presumably owing to the suicidal methylation of DNA which rendered it susceptible to the host endonuclease. The few clones in which pMP10 was established had lost DpnI . Loss of the plasmid after curing of the cell eliminated the methylase but did not restore DpnI . Although this loss of DpnI could result from spontaneous mutation, its relatively high frequency, 0.1% suggested that the loss was due to a regulatory shift.     

Interspecific plasmid transfer between Streptococcus pneumoniae and Bacillus subtilis

Summary The streptococcal plasmids pMV158 and pLS1, grown in Streptococcus pneumoniae, were transferred to Bacillus subtilis by DNA-mediated transformation. The plasmids were unchanged in the new host; no deletions were observed in 80 instances of transfer. Their copy number was similar to that in S. pneumoniae. Two B. subtilis plasmids, pUB110 and pBD6, could not be transferred to S. pneumoniae. Hybrid plasmids were produced by recombining the EcoRI fragment of pBD6 that confers Km^r with EcoRI-cut pLS1, which confers Tc^r. The simple hybrid, pMP2, was transferable to both species and expressed Tc^r and Km^r in both. A derivative, pMP5, which contained an insertion in the pBD6 component, expressed a higher level of kanomycin resistance and was more easily selected in S. pneumoniae. Another derivative, pMP3, which contained an additional EcoRI fragment, presumably of pneumococcal chromosomal DNA, could not be transferred to B. subtilis. Previous findings that monomeric plasmid forms could transform S. pneumoniae but not B. subtilis were confirmed using single plasmid preparations. Although plasmids extracted from either species were readily transferred to S. pneumoniae, successive passage in B. subtilis increased the ability of plasmid extracts to transfer the plasmid to a B. subtilis recipient. This adaptation was tentatively ascribed to an enrichment of multimeric forms in extracts of B. subtilis as compared to S. pneumoniae. A review of host ranges exhibited by plasmids of Gram-positive bacteria suggested differences in their ability to use particular host replication functions. The pMP5 plasmid, with readily selectable Km^r and Tc^r markers in both hosts, and with the potential for inactivation of Km^r by insertion in the Bg/II site, could be a useful shuttle vector for cloning in S. pneumoniae and B. subtilis.