Natural Diversity of Frankia Strains in Actinorhizal Root Nodules from Promiscuous Hosts in the Family Myricaceae

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N₂-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia.     

Purification and Characterization of NADH-Glutamate Synthase from Alfalfa Root Nodules

Glutamate synthase (GOGAT), a key enzyme in the pathway for the assimilation of symbiotically fixed dinitrogen (N₂) into amino acids in alfalfa (Medicago sativa L.) root nodules, was purified and used to produce high titer polyclonal antibodies. Purification resulted in a 208-fold increase in specific activity to 13 micromole per minute per milligram of protein and an activity yield of 37%. Further purification to near homogeneity was achieved by fast protein liquid chromatography, but with substantial loss of activity. Enzymic activity was highly labile, losing 3% per hour even when substrates, stabilizers, and reducing agents were included in buffers. However, activity could be partially stabilized for up to 1 month by storing GOGAT at −80°C in 50% glycerol. The subunit molecular weight of GOGAT was estimated at 200 ± 7 kilodaltons with a native molecular weight of 235 ± 16 kilodaltons, which suggested that GOGAT is a monomer of unusually high molecular weight. The pl was estimated to be 6.6. The K_m values for glutamine, α-ketoglutarate, and NADH were 466, 33, and 4.2 micromolar, respectively. Antibodies were produced to NADH-GOGAT. Specificity of the antibodies was shown by immunotitration of GOGAT activity. Alfalfa nodule NADH-GOGAT antibodies cross-reacted with polypeptides of a similar molecular weight in a number of legume species. Western blots probed with anti-GOGAT showed that the high GOGAT activity of nodules as compared to roots was associated with increased levels of GOGAT polypeptides. Nodule NADH-GOGAT appeared to be highly expressed in effective nodules and little if any in other organs.     

Identification of Frankia Strains by Direct DNA Hybridization of Crushed Nodules

A hybridization procedure was developed to identify Frankia strains inside actinorhizae by direct probing of crushed root nodules. The probe consisted of an indigenous cryptic plasmid. This well-conserved, 8-kilobase plasmid was detected in Frankia isolates that were very close taxonomically (they possessed a very high DNA sequence homology). The probe did not hybridize to the DNA of Frankia isolates which did not carry the plasmid. Endophyte DNA was extracted by a modification of a technique originally developed for the detection of plasmids in Frankia isolates. The hybridization procedure applied to nodules collected in a stand of alder permitted determination of a distribution map of the plasmid-bearing Frankia strains.     

Introduction of temperature-sensitive helper and donor plasmids into Bac-to-Bac baculovirus expression systems

In the baculovirus shuttle vector(bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome,and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive(ts) plasmid, p SIM6. Using the resulting ts helper plasmid p MON7124 ts and the ts donor plasmid p FB1ts-PH-GFP, a recombinant bacmid,b Ac WT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For b Ac WT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.     

Construction of In-Frame aroA Deletion Mutants of Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus by Using a New Temperature-Sensitive Plasmid

A temperature-sensitive (TS) plasmid was generated from the endogenous streptomycin resistance plasmid of Mannheimia hemolytica and used to engineer in-frame aroA deletion mutants of Mannheimia hemolytica, Pasteurella multocida, and Haemophilus somnus. TS replacement plasmids carrying in-frame aroA deletions were constructed for each target species and introduced into host cells by electroporation. After recovery in broth, cells were spread onto plates containing antibiotic and incubated at 30°C, the permissive temperature for autonomous plasmid replication. Transfer of transformants to selective plates cultured at a nonpermissive temperature for plasmid replication selected for single-crossover mutants consisting of replacement plasmids that had integrated into host chromosomes by homologous recombination. Transfer of the single-crossover mutants back to a permissive temperature without antibiotic selection drove plasmid resolution, and, depending on where plasmid excision occurred, either deletion mutants or wild-type cells were generated. The system used here represents a broadly applicable means for generating unmarked mutants of Pasteurellaceae species.     

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants

Transposon-insertion mutants with vir^? Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir^? nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir^? octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir^? octopine Ti plasmid mutations and vir^? nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO^? mutants produced rooty tumors on Kalanchoë tubiflora.     

Plasmid pCS1966, a New Selection/Counterselection Tool for Lactic Acid Bacterium Strain Construction Based on the oroP Gene, Encoding an Orotate Transporter from Lactococcus lactis

In this paper we describe the new selection/counterselection vector pCS1966, which is suitable for both sequence-specific integration based on homologous recombination and integration in a bacteriophage attachment site. This plasmid harbors oroP, which encodes a dedicated orotate transporter, and can replicate only in Escherichia coli. Selection for integration is performed primarily by resistance to erythromycin; alternatively, the ability to utilize orotate as a pyrimidine source in a pyrimidine auxotrophic mutant could be utilized. Besides allowing the cell to utilize orotate, the transporter renders the cell sensitive to 5-fluoroorotate. This sensitivity is used to select for loss of the plasmid. When expressed from its own promoter, oroP was toxic to E. coli, whereas in Lactococcus lactis the level of expression of oroP from a chromosomal copy was too low to confer 5-fluoroorotate sensitivity. In order to obtain a plasmid that confers 5-fluoroorotate sensitivity when it is integrated into the chromosome of L. lactis and at the same time can be stably maintained in E. coli, the expression of the oroP gene was controlled from a synthetic promoter conferring these traits. To demonstrate its use, a number of L. lactis strains expressing triosephosphate isomerase (tpiA) at different levels were constructed.     

Variation of the Linkage of Root Function with Root Branch Order

Mounting evidence has shown strong linkage of root function with root branch order. However, it is not known whether this linkage is consistent in different species. Here, root anatomic traits of the first five branch order were examined in five species differing in plant phylogeny and growth form in tropical and subtropical forests of south China. In Paramichelia baillonii, one tree species in Magnoliaceae, the intact cortex as well as mycorrhizal colonization existed even in the fifth-order root suggesting the preservation of absorption function in the higher-order roots. In contrast, dramatic decreases of cortex thickness and mycorrhizal colonization were observed from lower- to higher-order roots in three other tree species, Cunninghamia lanceolata, Acacia auriculiformis and Gordonia axillaries, which indicate the loss of absorption function. In a fern, Dicranopteris dichotoma, there were several cortex layers with prominently thickened cell wall and no mycorrhizal colonization in the third- and fourth-order roots, also demonstrating the loss of absorptive function in higher-order roots. Cluster analysis using these anatomic traits showed a different classification of root branch order in P. baillonii from other four species. As for the conduit diameter-density relationship in higher-order roots, the mechanism underpinning this relationship in P. baillonii was different from that in other species. In lower-order roots, different patterns of coefficient of variance for conduit diameter and density provided further evidence for the two types of linkage of root function with root branch order. These linkages corresponding to two types of ephemeral root modules have important implication in the prediction of terrestrial carbon cycling, although we caution that this study was pseudo-replicated. Future studies by sampling more species can test the generality of these two types of linkage.     

Method to integrate multiple plasmids into the mycobacterial chromosome

In order to create a system in which two independent plasmids can be integrated into a mycobacterial chromosome, a mycobacterial plasmid was constructed containing the phage attachment site attP from the mycobacteriophage L5 genome and additionally containing the bacterial attachment site, attB. This plasmid will integrate into the mycobacterial chromosome via recombination of the plasmid-borne attP site with the chromosomal attB site in the presence of a mycobacterial vector carrying the L5 integrase (int) gene. The integrated plasmid has a plasmid-borne attB site that is preserved and will accept the integration of additional mycobacterial plasmids containing the L5 attP site. This system should be useful in the construction of novel mycobacterial strains. In particular, this system provides a method by which several recombinant antigens or reporter constructs can be sequentially inserted into a mycobacterial strain and subsequently tested.     

Construction of pYGK,an Actinobacillus actinomycetemcomitans-Escherichia coli shuttle vector

A shuttle vector that is capable of replicating in Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) was constructed by modifying the Actinobacillus pleuropneumoniae (Ap) plasmid pYG53. A DNA fragment containing the Km^R gene was inserted into pYG53 to generate pYGK, which confers resistance to kanamycin in both Aa and Ec. By electroporation, Ec DH5α and 17 strains of Aa were transformed with pYGK with efficiencies ranging from 0.5 to 3 × 10⁶ colonies per μg of DNA. Plasmid pYGK exists at approx. 3–4 copies per cell in Ec. This plasmid will facilitate the genetic manipulation of Aa strains and the molecular analysis of virulence factors expressed by this organism     

Allelic retrieval: a scheme to facilitate the repeated isolation of a specific segment of the Bordetella pertussis chromosome

A plasmid vector was designed, constructed, and used for the repeated retrieval of the bvgA gene from a number of Bordetella pertussis strains that, due to mutations in this gene, exhibited interesting phenotypes regarding the regulation of virulence genes. The vector was used in a scheme called allelic retrieval that exploits two cross-overs between cloned plasmid and native chromosomal sequences flanking the bvgA gene. This scheme is very similar to allelic exchange through the use of plasmid suicide vectors, but in the case presented here, the non-replicating plasmid that has received the chromosomal gene is recovered, rather than being allowed to be lost due to segregation. Incorporation of the counterselectable sacB gene of Bacillus subtilis in place of the plasmid copy of bvgA allows selection, after recovery in Escherichia coli, for only those plasmids that have retrieved the chromosomal bvgA gene. The validity of this approach was demonstrated by the retrieval of bvgA alleles with distinctive physical markers, as well as by the reintroduction of retrieved bvgA alleles to demonstrate that they conferred the expected phenotypes. It is expected that this approach will be applicable to the analysis of other genes in other bacterial species.     

Electroporation-mediated transformation of the ovine footrot pathogen Dichelobacter nodosus

Studies on the potential virulence genes of the ovine footrot pathogen Dichelobacter nodosus have been hindered by the lack of a genetic system for this organism. In an attempt to accomplish the transformation of D. nodosus cells, we constructed a plasmid that contained part of a native D. nodosus plasmid and carried a tetracycline resistance gene that was located between the D. nodosus rrnA promoter and terminator. This plasmid was used to transform several D. nodosus strains to tetracycline resistance. Analysis of two independent transformants from each parental strain showed that in nearly all of these derivatives, the plasmid was not replicating independently, but that the tetracycline resistance gene had inserted by homologous recombination into one of the three rrn operons located on the chromosome. In most of the transformants, double reciprocal crossover events had occurred. These results are highly significant for genetic studies in D. nodosus and for footrot pathogenesis studies, since by using reverse genetics it will now be possible to examine the role of putative D. nodosus-encoded virulence genes in the disease process.     

Construction of an alkaline phosphatase fusion-generating transposon,mTn10phoA

We constructed a derivative of the mini-transposon mTn10 that generates translational fusions to the phoA gene from Escherichia coli and carries the Km^R determinant from Tn5. This new transposon, mTn10phoA, is carried on a mobilizable plasmid with both selectable and counterselectable markers. The plasmid carrying mTn10phoA was introduced into Legionella pneumophila. Southern hybridization analysis indicated that the mTn10phoA insertions were randomly distributed     

Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of α-proteobacteria.     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^? strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.