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Applied and Environmental Microbiology, February 2004, p. 991-998, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.991-998.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Selection of Unusual Actinomycetal Primary ⁷⁰ Factors by Plant-Colonizing Frankia Strains

Céline Lavire, Didier Blaha, and Benoit Cournoyer^*

Research Group on Opportunistic Pathogens and the Environment, Ecologie Microbienne (Center for Microbial Ecology), UMR CNRS 5557-Université Claude Bernard-Lyon 1, USC INRA 1193, 69622 Villeurbanne Cedex, France

Received 4 June 2003/ Accepted 31 October 2003

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Functional adaptations of ⁷⁰ transcriptional factors led to the emergence of several paralogous lineages, each one being specialized for gene transcription under particular growth conditions. Screening of a Frankia strain EaI-12 gene library by ⁷⁰ DNA probing allowed the detection and characterization of a novel actinomycetal primary (housekeeping) ⁷⁰ factor. Phylogenetic analysis positioned this factor in the RpoD cluster of proteobacterial and low-G+C-content gram-positive factors, a cluster previously free of any actinobacterial sequences. ⁷⁰ DNA probing of Frankia total DNA blots and PCR screening detected one or two rpoD-like DNA regions per species. rpoD matched the conserved region in all of the species tested. The other region was found to contain sigA, an alternative primary factor. sigA appeared to be strictly distributed among Frankia species infecting plants by the root hair infection process. Both genes were transcribed by Frankia strain ACN14a grown in liquid cultures. The molecular phylogeny of the ⁷⁰ family determined with Frankia sequences showed that the alternative actinomycetal factors and the essential ones belonged to the same radiation. At least seven distinct paralogous lineages were observed among this radiation, and gene transfers were detected in the HrdB actinomycetal lineage.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
⁷⁰ factors play a key role in gene expression by binding reversibly to eubacterial DNA-dependent RNA polymerase to form the holoenzyme and enabling the promoter-specific attachment of the complex. The holoenzyme then can drive the synthesis of mRNA (but the process could be regulated by several transcriptional repressors [27] and activators [19]). Through evolution, selective pressures seem to have favored the emergence of several paralogous lineages of ⁷⁰, and these factors became a central component of bacterial adaptability to changing environments. As an illustration, different ⁷⁰ factors were found to be involved in stress responses, the production of extracytoplasmic factors, and cellular differentiation processes, such as sporangium formation. These findings resulted in the discovery of several genes per genome encoding ⁷⁰ factors. Among the actinomycetes, 13 putative sigma factors were detected in the complete genome sequence of Mycobacterium tuberculosis strain H37Rv (5), and 65 were detected in the genome sequence of Streptomyces coelicolor (1). ⁷⁰ factors were divided into four groups according to sequence similarities and promoter recognition specificities (17): group 1 includes the essential sigma factors, termed the "primary sigma factors," which are involved mainly in exponential growth; group 2 includes the stationary-phase and alternative actinomycetal sigma factors; group 3 includes the factors involved in sporangium development, flagellin synthesis, and the heat shock response; and group 4 includes the extracytoplasmic function sigma factors. These factors were shown to have affinities for different promoter regions and to be regulated by distinct processes. However, they are in competition for the same DNA-dependent RNA polymerase core enzyme (20).

In the above classification, the grouping of stationary-phase and alternative actinomycetal sigma factors (group 2) could be misleading, giving the impression of some sort of common functional features (which are not demonstrated). Phylogenetic analysis of groups 1 and 2 showed that the gram-negative stationary-phase factors formed a tight group apart from the alternative actinomycetal sigma factors, which seemed more closely related to the primary factors (13). These groupings are revised in this study. It is noteworthy that despite the number of sigma factors reported among the actinomycetes (and the names used to designate the primary sigma factors—hrd meaning "homologs of Escherichia coli rpoD"), sequences belonging to the RpoD lineage, which are essential for growth of the Proteobacteria and Firmicutes (low-G+C-content gram-positive organisms), never were detected (13). This observation was interpreted as evidence of the nonmonophyletic origins of the low- and high-G+C-content gram-positive bacteria (13).

Recently, Blaha and Cournoyer reported the characterization of the first Frankia sigma factor, SigA (2). Frankia species are pleiomorphic nitrogen-fixing actinomycetes that can interact with woody dicots and lead to the formation of root nodules. This sigma factor belongs to a novel lineage of sigma factors which is separate from the other lineages but is related to Streptomyces alternative sigma factors, such as HrdA. The sigA gene was isolated from a gene library of a Frankia strain (ACN14a) that infects alder trees. Considering the number of sigma factors found per actinomycetal genome so far, it was surprising that a single member of the ⁷⁰ family was recovered in the course of the latter study—especially a factor that does not belong to the lineage of essential actinomycetal primary sigma factors (HrdB homologs).

Here, the Frankia ⁷⁰ gene family was further investigated by ⁷⁰ DNA probing of a gene library of a genomic species that infect Elaeagnus plants. This study identified a novel Frankia ⁷⁰ gene which was never recorded among the other actinobacteria. Its DNA sequence, deduced protein, transcription, distribution, and phylogenetic position in the ⁷⁰ family were analyzed. Comparative studies were performed with Frankia sigA. The phylogenetic analyses performed during this work clarified several ambiguities in the evolutionary trends of this gene family.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strains and DNA extractions.
The following strains were used in this study: ACN1^AG (16) and ACON24d (30) of Frankia alni (Alnus infectivity group; colonization by the root hair infection [RHI] process [4]); ACN14a (23) and ArgP5^AG (24) (representative strains of two genomic species of the Alnus infectivity group; RHI); CeD (9), CcI3 (33), and ORS020607 (9) (strains of genomic species of the Casuarina infectivity group; RHI); Flec (A. M. Domenach and Y. Hammad, personal communication), HRX401a (25), and EaI-12 (10) (strains of genomic species of the Elaeagnus infectivity group; direct root penetration and intercellular migration [IP] process [22]); HR2714 (this study) (infective for Hippophae; IP); and D11 (12) (isolated from Alnus but infective for Elaeagnus). Growth conditions for Frankia strains and DNA extraction procedures were those described by Simonet et al. (31).

PCR and RT-PCR amplifications.
All PCR primers used in this study are described in Fig. 1. PCR amplifications (50 µl) were performed according to the instructions of the Taq polymerase manufacturer (Gibco BRL/Life Technologies, Cergy Pontoise, France). PCR cycles with total DNA were as follows: 95°C for 6 min; three cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 30 s; 42 cycles of 95°C for 30 s, selected annealing temperatures for 30 s (Fig. 1), and 72°C for 30 s; and 7 min at 72°C. Extraction of RNA from 7-, 8-, and 12-day-old Frankia ACN14a liquid cultures in BAP (22a) supplemented with N (50 ml) was performed as described by Maréchal et al. (21). This extraction includes DNA digestion with DNase. The efficacy of the DNase treatments was verified by PCR with 100 ng of RNA and the bfr gene as a control target (bacterioferretin) (21). cDNAs were prepared from 1 µg of RNA by using Moloney murine leukemia virus reverse transcriptase (RT; Gibco BRL). PCR screening of the cDNAs with primers Sig1.5 and Sig2.5 and with primers Sig1.6 and Sig2.4 (Fig. 1 shows the sequences) was performed as described above at an annealing temperature of 60°C.

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FIG. 1. Nucleotide sequence alignment for Frankia EaI-12 rpoD and Frankia ACN14a sigA genes. Dots indicate identities. Gaps were introduced to match the sequences properly. Putative start codons are underlined; stop codons are indicated by asterisks. A putative Frankia Shine-Dalgarno sequence is indicated by double underlining. Arrows indicate the primers used in this study. Annealing temperatures used for PCR amplifications were as follow: 58°C for primers Sig1.1, Sig1.3, Sig1.4, Sig1.7, Sig1.8, Sig2.2, and Sig2.3; and 60°C for Sig1.2, Sig1.5, Sig1.6, Sig2.1, Sig2.5, and Sig2.4.

DNA subcloning and sequencing, EMBL3 gene library, and DNA blot analyses.
DNA manipulations were performed as described by Sambrook et al. (29). Restriction enzymes were used as recommended by the manufacturer (Gibco BRL). PCR fragments were cloned into pGEM-T Easy as instructed by the manufacturer (Promega Corp., Lyon, France). Subcloning-efficient DH5 competent cells were used for cloning as recommended by the manufacturer (Gibco BRL).

The EaI-12 EMBL3 gene library was built as recommended by Stratagene (Amsterdam, The Netherlands). It was transferred to nylon membranes as described by Sambrook et al. (29). A total of 20,000 PFU, corresponding to about 30 times the Frankia genome, were screened with a radioactively labeled Sig1.7-Sig2.1 PCR fragment amplified from Frankia ACON24d total DNA (named the ⁷⁰ DNA probe). Membranes were hybridized and washed at 65°C by following the recommendations for GeneScreen (Perkin Elmer Life Sciences, Inc., Boston, Mass.). Autoradiography was performed as recommended by Amersham Pharmacia Biotech (Orsay, France). EMBL3 DNA extraction of hybridizing PFU was performed with a Wizard Lambda Preps purification system (Promega). Blot analysis of SalI digests of EMBL3 library clone DNAs was performed with the ⁷⁰ DNA probe. One phage, 8.51 (insert of about 13 kb), was selected for further characterization. A 8.51 1.3-kb SalI hybridizing fragment was subcloned into pBluescript KS(-) (OZYME, Schwabach, Germany), giving pFEQ102, and sequenced. DNA fragments were extracted and purified from agarose gels by using a Qiaquick gel extraction kit (Qiagen S.A., Courtaboeuf, France).

DNA sequencing of pBluescript KS(-) and pGEM-T derivatives was performed by using Genome Express Services (Meylan, France) with M13 forward (M13F) and M13 reverse (M13R) universal primers. Sequencing of 8.51 and pFEQ102 was completed with the primers shown in Fig. 1.

Blot analysis of SmaI and KpnI digests of Frankia total DNA with the ⁷⁰ DNA probe was performed by following the recommendations for GeneScreen. Hybridizations and washes were carried out at 58°C.

Phylogenetic analyses.
The GCG package of the University of Wisconsin (8) and Frame plot version 2.3.2 (14) were used to find coding sequences and to determine their corresponding deduced putative proteins. BLAST searches were run at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast/) to detect DNA and protein identities or similarities shared with accession numbers in international databases. Multiple alignments were computed by using CLUSTAL W (32). Distances between sequence pairs, inferred phylogenetic trees, and bootstrap values were all computed by using the phylo_win graphic tool (11). Phylogenetic trees were built by using the neighbor-joining (NJ) method (28). Evolutionary distances between DNA sequence pairs were estimated by using the Kimura two-parameter model (15).

Nucleotide sequence acession numbers
DNA sequence of the Frankia sp. strain EaI-12 rpoD locus reported here has the GenBank accession no. AY423462. PCR-amplified and -sequenced Frankia rpoD and sigA sequences have the following GenBank accession numbers: AY423463, AY423464, AY423465, AY423466, AY423467, AY423468, AY423469, AY423470, AY423471, AY423472, and AY423473.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Characterization of a novel primary actinobacterial ⁷⁰ lineage.
⁷⁰ DNA probing of the EMBL3 Frankia strain EaI-12 gene library and subsequent DNA subcloning of the hybridizing regions allowed the recovery of a 1.3-kb fragment, which was fully sequenced. BLASTX analysis of this DNA sequence detected an overlap between a hypothetical deduced protein of 279 amino acids and S. aureofaciens HrdB sigma factor (the essential actinomycetal factor), which showed 75% identity and 87% similarity beginning at position 248 of HrdB. This fragment contains a partial coding sequence, which was further sequenced by using EMBL3 clone 8.51. A 1,770-nucleotide sequence was obtained (accession no. AY423462).

Test code analysis of this Frankia EaI-12 DNA sequence was performed to identify putative open reading frames (ORFs). A probable coding sequence was detected from positions 1 to 1306 and is shown in Fig. 1. An ORF was detected from positions 77 (GTG) to 1306 (TAG). This ORF (named orfB; its deduced protein is named OrfB) is 1,230 bp long and encodes a putative protein of 410 amino acids. A typical Frankia ribosome-binding site is observed 10 bp upstream of the start codon. BLASTP searches detected similarities between OrfB and bacterial ⁷⁰ factors. A BLAST two-sequence analysis between OrfB and S. griseus HrdB showed two regions of similarity: one with 35% identity from positions 10 to 76 of HrdB and matching positions 19 to 85 of EaI-12 OrfB and one with 72% identity from positions 214 to 514 of HrdB and matching positions 108 to 409 of OrfB. A BLAST two-sequence analysis showed 63% identity between the Frankia EaI-12 orfB sequence and Frankia ACN14a sigA (2) (Fig. 1). All of the conserved motifs (4 regions and 10 subregions) described by Lonetto et al. (17, 18) were observed in the Frankia EaI-12 OrfB sequence.

Phylogenetic inferences.
The close similarity of OrfB and HrdB, described above, led us to investigate further the phylogenetic position of OrfB in the group 1 and 2 ⁷⁰ factors. A multiple alignment of representative protein sequences from these groups was generated. Phylogenetic distances between all pairs of sequences were computed, and an NJ phylogenetic tree was constructed (Fig. 2). The Mycobacterium SigF and Bacillus subtilis RpsB sequences were used as outgroups. The inferred NJ tree (Fig. 2) clearly is divided into two parts: cluster I (supported by 91% of the bootstrap replicates), grouping the main primary sigma factors and the alternative actinomycetal ones; and cluster II, grouping the proteobacterial stationary-phase factors. These phylogenetic groupings are not in perfect agreement with those defined by Lonetto et al. (17) and suggest that the groupings of Lonetto et al. should be amended accordingly. From now on, we thus will consider group 1 as being a lineage of primary factors including the essential ones, such as RpoD and HrdB, as well as the alternative actinomycetal primary factors. This modification leads to group 2 excluding the alternative actinomycetal factors included in the newly defined group 1.

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FIG. 2. NJ phylogenetic tree for group 1 and 2 70 factors (amino acid sequences). The sequences were from this work or from Lonetto et al. (17) or were retrieved from the GenBank database. A total of 197 sites (of which 162 were informative) were analyzed. Distances are proportional to evolutionary divergences expressed in substitutions per 100 sites. Some branch lengths are indicated on the tree branches. Bootstrap values higher than 85% are given in ovals. The asterisk indicates a significant bootstrap value (97%) observed for this cluster with a slightly different data set (2). Accession numbers are shown.

In cluster I (group 1 of essential and alternative actinomycetal primary ⁷⁰ factors), eight well-resolved phylogenetic clusters or lineages are observed (Fig. 2): A, the Streptomyces HrdA alternative ⁷⁰ lineage (for 100% of the bootstrap replicates); B, the MysB-SigB alternative ⁷⁰ lineage found in Mycobacterium and Brevibacterium (for 100% of the bootstrap replicates); C, the HrdB essential primary ⁷⁰ lineage found in Streptomyces, Renibacterium, Corynebacterium, Brevibacterium, and Mycobacterium (for 100% of the bootstrap replicates); D, the lineage of essential primary sigma factors found in Firmicutes (low-G+C-content gram-positive bacteria) and including the Frankia OrfB sequence (for 92% of the bootstrap replicates); E, the RpoD lineage of essential sigma factors found in Proteobacteria (for 90% of the bootstrap replicates); F, the Frankia SigA lineage (for 100% of the bootstrap replicates); G, the Streptomyces HrdC lineage; and H, the Streptomyces HrdD lineage (for 100% of the bootstrap replicates). Lineages D and E (corresponding to Proteobacteria and Firmicutes essential primary ⁷⁰ sequences) are grouped but not at a significant level of bootstrap values. However, the bootstrap values were found to be significant when a slightly different data set was used (2). A single lineage (RpoD) is observed in the Proteobacteria. Lineages A, F, and H seem to be genus specific. All lineages have deep branches. In cluster II (stationary-phase factors) of the ⁷⁰ NJ tree, two main groups are observed: one grouping the sequences from the -proteobacteria (supported by 98% of the bootstrap replicates) and the other grouping the sequences from the ß-proteobacteria (supported by 92% of the bootstrap replicates).

It can be inferred from the various analyses of the ⁷⁰ family (13, 17, 18, 26; this work) that group 1 and 2 factors share a common ancestral sequence. A duplication event appears to have led to the emergence of these two paralogous lineages. After this duplication, orthologous evolution of the proteobacterial stationary-phase factors of group 2 seems to have proceeded. However, this was not the case for the group 1 lineage. A major amplification appears to have occurred, leading to at least seven paralogous lineages (A, B, C, D/E, F, G, and H) (Fig. 2). This conclusion was inferred from a comparison of the lineages in group 1 and those of an rrn NJ tree derived from sequences of the taxons (or closest relatives) used in this study. This comparison suggested that the group 1 radiation occurred early in the evolution of bacteria, prior to the diversification of eubacteria into their well-recognized phylogenetic groups (e.g., Proteobacteria, Firmicutes, and Actinobacteria). Selective pressures seem to have favored the deletion of most lineages from the Proteobacteria and Firmicutes, but the Actinobacteria appear to have conserved most of them. Following this amplification of primary factors, orthologous evolution appears to have proceeded in each lineage. The only cases of genetic transfer and duplication are restricted to lineage C. Two subgroups are observed in this lineage: one containing Mycobacterium, Brevibacterium, and Corynebacterium sequences and one containing Streptomyces and Renibacterium sequences (both for 100% of the bootstrap replicates). These subgroups are not in accord with the rrn NJ tree (Fig. 3). Renibacterium and Brevibacterium are recognized genera of the Micrococcineae, and their rrn sequences form a significant group in the rrn NJ tree (for 95% of the bootstrap replicates), apart from the sequences of Streptomyces and Corynebacterineae and inside the cluster of sequences from Actinobacteria (for 100% of the bootstrap replicates). The most likely explanation for this discrepancy is lateral gene transfer of lineage C determinants, followed by some sort of genome reorganization leading to the selection of the newly acquired copy and the deletion of the original one. In lineage C, a duplication event led to the finding of two closely related sequences in the S. griseus genome (Fig. 2).

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FIG. 3. rrn (16S ribosomal DNA) NJ phylogenetic tree from bacteria or their closest relatives harboring reported group 1 or 2 70 factors (Fig. 2). A total of 839 sites (of which 303 were informative) were analyzed. All sequences were retrieved from GenBank. Vertical distances are proportional to phylogenetic distances. Horizontal distances are for clarity only. Bootstrap values of higher than 86% are given in ovals. See the text for details. The full bacterial names are given in Fig. 2. Accession numbers are shown.

Functional analysis of the lineage C HrdB gene of S. coelicolor (3) showed this lineage to contain genes encoding the essential ⁷⁰ primary factors of the actinomycetes. However, a lineage C homolog was not detected in Frankia. Instead, the OrfB factor (characterized here) was found and shown to group with the RpoD sequences of the D/E cluster. Accordingly, the Frankia EaI-12 OrfB factor was named RpoD. This position in the ⁷⁰ NJ tree is a strong argument for considering this lineage the essential ⁷⁰ primary factor lineage in Frankia. This notion represents a major variation in the evolutionary history of actinobacterial ⁷⁰ primary factors. Specialized selective pressures that might be related to the symbiotic properties of Frankia appear to have favored the deletion and selection of distinct lineages in these actinomycetes. This situation is true not only for the essential ⁷⁰ factor but also for the alternative SigA factor. This factor was observed only in three genomic species of Frankia (see below). It is noteworthy that the positioning of Frankia OrfB in the RpoD lineage gives a topology to this subgroup which matches that of the rrn tree. Without the OrfB sequence, the essential primary ⁷⁰ factor of the Actinobacteria would appear to have diverged prior to the diversification of the sequences of the Firmicutes (low-G+C-content gram-positive bacteria) from those of the Proteobacteria—a situation that was previously observed by Gruber and Bryant (13) but that does not appear to represent the relationships among gram-positive bacteria.

Distributions of rpoD and sigA in Frankia strains.
Several PCR primers were defined from conserved and variable regions observed in the BLAST two-sequence alignment of the ACN14a and EaI-12 ⁷⁰ sequences (Fig. 1). These primers were used to PCR amplify ⁷⁰ sequences from total DNAs isolated from Frankia strains that infect Alnus, Casuarina, Hippophae, or Elaeagnus. sigA primers Sig1.1 and Sig2.1 were successfully used to amplify a 1-kb fragment from strain ACON24d but failed to amplify any other DNA target from the total DNAs tested. Primers Sig1.3 (rpoD) and Sig2.1 (sigA) PCR amplified 800-bp fragments from total DNAs of strains ACN1^AG, Flec, HRX40Ia, HR2714, and D11. Primers Sig1.4 (rpoD) and Sig2.2 (sigA) PCR amplified 700-bp fragments from DNAs of strains ORS020607, CeD, CcI3, and ArgP5^AG. All of these PCR products were cloned in pGEM-T and sequenced (Fig. 4 shows the accession numbers). Apart from the ACON24d PCR product (Sig1.1 and Sig2.1 primers), all sequences were found, by BLAST two-sequence analysis, to be closer relatives of the EaI-12 rpoD gene sequence than the ACN14a sigA gene sequence. However, a XhoI digest of ORS020607 PCR products showed the presence of distinct products under a single electrophoretic signal. This variability was not observed among the cloned PCR fragments. A Southern blot analysis of SmaI-digested ACN14a, ORS020607, CeD, and ACN1^AG total DNAs showed two DNA regions in these genomes hybridizing with the ⁷⁰ DNA probe described above (data not shown). Two novel primers, Sig1.6 (sigA) and Sig1.5 (rpoD), thus were designed. These primers were successfully used, in combination with primer Sig2.2, to amplify rpoD-like PCR products with strain ACN14a total DNA or sigA-like PCR products with strain ORS020607 total DNA. These PCR products were sequenced (Fig. 4 shows the accession numbers). PCR with the sigA-specific primer Sig1.6 used in combination with primer Sig2.1, Sig2.2, or Sig2.4 failed to amplify sigA from total DNAs of strains that infect Elaeagnus or Hippophae.

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FIG. 4. NJ phylogenetic tree for Frankia 70 primary gene sequences. Sequences were obtained during this work or were retrieved from GenBank. A total of 604 sites (of which 199 were informative) were analyzed. Distances are proportional to evolutionary divergences expressed in substitutions per 100 sites. They are indicated on the tree branches. Bootstrap values higher than 85% are given in ovals. IP, implying direct root penetration and intercellular migration; RHI, implying colonization by the root hair infection process. Accession numbers are shown.

A multiple alignment with all of the Frankia ⁷⁰ DNA sequences available was generated and used to estimate phylogenetic distances between pair of sequences. An NJ phylogenetic tree was inferred from these distances. The NJ tree (Fig. 4) is divided into two major clusters (supported by 100% of the bootstrap replicates): one grouping the sigA sequences from Frankia ACN14a, ACON24d, and ORS020607 and one grouping the rpoD sequences from the Frankia strains studied. In the sigA cluster, the sequences obtained for the strains isolated from Alnus form a distinct lineage (for 100% of the bootstrap replicates). The few lineages in the Frankia sigA cluster show an organization in agreement with that of the rpoD sequences from the same strains. Despite several attempts, the Frankia sigA lineage could be detected only in Frankia strains or genomic species that were strictly infective through RHI. This factor thus could play a role in the colonization of plants specialized for this process (e.g., Alnus, Myrica, and Casuarina). However, transcriptional analysis of sigA and rpoD with Frankia strain ACN14a showed both genes to be transcribed constitutively in pure cultures (see below).

In the rpoD cluster, two groups, which match Frankia host specificities, can be distinguished (supported by 100% of the bootstrap replicates): a cluster containing sequences from Frankia strains isolated from Elaeagnus and Hippophae nodules—infective through the IP process; and a cluster containing sequences from Frankia strains isolated from Alnus and Casuarina—infective through the RHI process. The sequence of atypical strain D11 is positioned outside these two clusters. In the rpoD cluster of the RHI strains, three lineages can be distinguished: one grouping sequences from strains that infect Casuarina (for 100% of the bootstrap replicates), one grouping sequences from strains ACN1^AG and ACN14a that infect Alnus (97% of the bootstrap replicates), and a lineage corresponding to the sequence from strain ArgP5^AG. The good agreement between these phylogenetic groups or lineages and Frankia host specificities is in accord with the results of previous studies with rrs, glnII, and nifHD data sets (6, 7). The partial rpoD sequences from the Frankia strains infective for Casuarina are identical (100% identity). The sequences from Frankia strains that infect Elaeagnus and Hippophae show 99% identity. The sequences from Frankia strains that infect Alnus show 95% identity.

rpoD and sigA mRNA analyses.
Total RNA was isolated from Frankia ACN14a cells that had been grown for 7, 8, and 12 days in liquid cultures and reverse transcribed (by using 1 µg of the DNA-free RNA obtained). Primers Sig1.6 and Sig2.4 and primers Sig1.5 and Sig2.5 were used to selectively amplify Frankia sigA and rpoD fragments, respectively. PCR amplification was observed for both sets of primers and for all incubation times, indicating that both genes were transcribed under the conditions used (Fig. 5). AvaI enzymatic digestion confirmed the identity of the PCR products; this restriction generated, as expected, fragments of 170 and 82 bp for sigA and of 210 and 35 bp for rpoD (Fig. 5).

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FIG. 5. RT-PCR analysis of Frankia ACN14a sigA and rpoD. Lanes A, C, and E, ropD RT-PCR products obtained from 7-, 8-, and 12-day- old liquid cultures, respectively. Lanes B, D, and F, sigA RT-PCR products from 7-, 8-, and 12-day-old liquid cultures, respectively. Lanes G and H, AvaI digests of rpoD and sigA RT-PCR products, respectively. PCR of 100 ng of DNase-treated Frankia RNA with the bfr, rpoD, or sigA primer did not yield any products. The lane between lanes F and G shows SmartLadder (Eurogentec).

The next step in these studies will be to characterize the genes or promoters controlled by the encoded RpoD and SigA factors and to try to understand the selective pressures that led to their conservation in Frankia.

 
We thank C. Monnez for technical assistance at various stages of this work. We thank J. Maréchal for sharing technical expertise on Frankia mRNA extractions.

We thank the CNRS and UCB-Lyon 1 for funding parts of this research.

 
* Corresponding author. Mailing address: Ecologie Microbienne, UMR CNRS 5557-Université Claude Bernard-Lyon 1, USC INRA 1193, 43 Bd. 11 Novembre 1918, BÂt. Gregor Mendel (741), 5th Floor, F-69622 Villeurbanne Cedex, France. Phone: 33 (0) 4 72 43 14 95. Fax: 33 (0) 4 72 43 12 23. E-mail: cournoye{at}biomserv.univ-lyon1.fr.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Applied and Environmental Microbiology, February 2004, p. 991-998, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.991-998.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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