Results and Discussion

3.1. A deletion within the tts gene cluster affects symbiosis in a host specific manner. The known tts gene clusters of rhizobia have at least 17 genes in common (Figure 1). To analyze their importance for symbiosis, we created the deletion derivative A136 that lacks several open reading frames. Compared to the wild type, nodulation with strain A136 was clearly delayed on Macroptilium atropurpureum and to a lesser extent on soybean (Figure 2). No nodulation defect was observable with Vigna unguiculata. Twenty days after inoculation, M. atropurpureum infected with strain A136 still had fewer nodules, however of increased size. Despite the fact that fewer nodules were induced on M. atropurpureum, acetylene reduction was hardly impaired on the basis of acetylene reduced per plant (data not shown). Hence, we observed an increase of nodule dry weight with the mutant strain.

M. loti

M. loti

ORFs encoding secreted proteins in NGR234 fusion to the uidA reporter gene

O ORFs specific to one strain besides y4vP its box

ORFs encoding secreted proteins in NGR234 fusion to the uidA reporter gene

O ORFs specific to one strain besides y4vP its box

Figure 1. Comparison of type III secretion gene clusters. Conserved regions are connected by shaded areas. The arrow points to the newly identified nop A gene that is conserved in all three strains.

wt A136 wt A136 wt Ä136

G. max M. atropurpureum V. unguiculata

Figure 2. Nodulation phenotype of mutant A136 (Figure 1). Numbers inside the columns indicate the quantity of plants used in the assay.

3.2. The nod box preceding y4xl (ttsA) is regulated by nodW and the nodD region. It is known that the common nod genes, which are preceded by a nod box, are activated by nodW and nodDl. Because y4xl (UsA) is also preceded by a nod box (Figure 1), we expect it to be regulated by nodW and nodDl as well. To test this, we constructed a transcriptional fusion with the uidA reporter gene. The fusion was inserted into the chromosome of the wt and different mutant strains. As predicted, expression was inducible by genistein in the wild-type background (Figure 3). The expression pattern did not change in the y4xl (ttsA) mutant, indicating that this gene is not involved in the regulation of the nod box promoter. However, no induction was observed in the nodW or the nodD mutant. Thus, either one of the regulatory systems is acting through the other system or both systems are acting at the same promoter. We favor the last idea due to the aforementioned similarity of the nod box with that in the common nod gene region. The Y4xl (TtsA) protein exhibits similarity to members of the two component regulatory systems. Furthermore, we found that it is involved in the regulation of a nolU-lacZ fusion. Because of this, we believe that Y4xl (TtsA) acts in a regulatory cascade downstream of NodW and NodDl as transcriptional activator of the tts cluster. Therefore, we suggest to change the designation of y4xl to ttsA (type three secretion).

wt y4xl nodW nodD2DlnolA~

Figure 3. Activity of a transcriptional nod box-uidA fusion in the wild type and mutant strains. Values are from at least five independent cultures.

3.3. Genes in the tts cluster are preceded by a common motif. If TtsA is the major activator of the genes in the tts cluster, then one might expect to find common sequence motifs upstream of transcription units. The result of a corresponding analysis is summarized in Figure 4. Upstream regions of all three rhizobial strains have significant similarity. The rhcCl-nolB intergenic regions exhibit a higher similarity than the remaining upstream sequences. In the alignment, we did not include the sequence of the upstream regions of nopX and nolB of S. fredii. The sequence of this strain (accession number Ay034152) is almost identical to the sequence of NGR234 and is invariant in the conserved elements. In S. fredii, however, the transcriptional start sites of nopX and nolB have been determined (Kovacs et al. 1995). They are located 11 bp and 12 bp downstream of the conserved elements, respectively. Therefore, it is likely that the conserved sequences serve as promoter elements. Nevertheless, the impact of the conserved nucleotides on the expression level remains to be elucidated.

Bjap rhcCl 90 bp - TCCGTCAGGTTTTCGTCAGCTCGGCAGCCTA - 49 bp nolB M.lotl rhcCl 80 bp - TCAGTCAGCTTGTCGTCAGCTCGGCCACCTA - 71 bp nolB NGR234 rhcCl 85 bp - GTAGTCAGCGTGTCGTCAGCTCGCCTCGCTA - 39 bp no IB Consensus GTCAG T TCGTCAGCTCG C CTA

Bjap

AGGCTCGTCAGCTTTTCGAAAGCTAGCGCCCCTA -

64

bp

nop A

M. loti

AGACTCGTCAGGTTCTCGAAAGCTCCTGCTCGTA -

446

bp

nopA

NGR2 34

ACAATTGTCAGCTTTTCGAAAGCTGGAGCTCATA -

410

bp

nop A

Bjap

ATCATCGTCAGCTTTTCGACAGGTGTTCGGGCTA -

221

bp

id205

NGR234

CTGATTGTCAGCTTCTCGAAAGGTATGTCTCTTA -

261

bp

nopL

Bjap

CCGATCGTCAGCTTTTCGAAAGCTAAAGCCCCCA -

109

bp

nopL

Bjap

GCGAT-GTCAGGTTTTGGAAAGCAAACGTGAGTA -

36

bp

id274

M. loti

GAACTCGTCAGTTTACCGAAAGCTAAACCGCTCA -

100

bp

nopX

NGR2 34

AGCCTCGTCAGTTTCTCGAAAGCTAAACCGCTCA -

189

bp

nopX

Consensus TcGTCAG TT TCGAAAGcTa c c tA

c tts box consensus tcGTCAGcTT TCGaaAGcT c c tA

Consensus TcGTCAG TT TCGAAAGcTa c c tA

c tts box consensus tcGTCAGcTT TCGaaAGcT c c tA

Figure 4. Alignment of putative promoter regions within the tts gene cluster. The numbers indicate the distance from the putative start codon. The upper part denotes the intergenic region between the divergently oriented nolB and rhcCl genes. Nucleotides that are conserved in all regions are underlined. Nucleotides that are conserved in all sequences except one are printed in capital letters. Lower case letters show the positions that are conserved in at least seven upstream regions.

3.4. The tts gene cluster contains a so-far unidentified conserved ORF that might encode a secreted protein. Reanalysis of the nucleotide sequence of all three rhizobial strains revealed the presence of a conserved ORF upstream of y4yQ (Figures 1 and 5). All are of comparable size. Similarity on the amino acid level is highest between the proteins of NGR234 and M. loti. In an earlier work, we determined the N-terminal amino acid sequence of an exported protein of B. japonicum. The obtained sequence AVDNVGG fits nicely to the N-terminal end of the deduced gene product of the new ORF. We suggest to name the gene nopA (nodulation outer protein) in line with the nomenclature suggested by Marie et al. (2001).

B j ap MA.VDNVG----GNGGAAG----AQQTGDSGFQNQMAEFERVSQKVQAQAVAMRRITTELS

NGR234 MSKIGTLTSAVGAGAAAGQNVAAKGAGAAAFQAQIAELAAVSAEATARSMLLRTVTTELQ

I I I I I I I I I I I II I I I II I I I I I I I I II I I I I II I I

M. loti MSKAGTGTSAAGTGTSVATDVGTRVAGKAAFEAQIADLTAASLEATARSIQLRTVTTNLN

Bjap SEKKVADERVQ 63

II I I I I I I NGR234 TTKKAADERVQ 71

Figure 5. Alignment of NopA proteins. The amino acid sequence that was obtained by N-terminal sequencing of an extracellular protein of B. japonicum is underlined.

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