42 Mb from the well-characterized Hrc-Hrp1 T3SS Q-VD-Oph in vitro cluster in the main chromosome. Both clusters are located on DNA segments with GC content similar to their neighbouring areas. No sequences associated with HrpL-responsive promoters (characteristic for the regulation of the Hrc-Hrp1
operons in P. syringae pathovars) were found in the T3SS-2 gene cluster  indicating a different way of regulation from the Hrc-Hrp1 selleck system. The ORF PSPPH_2539 that resides between the core genes and the hrpK homolog PSPPH_2540, codes for a hypothetical transcription regulator (Figure 4, 5). No t RNA genes, however, have been found in the vicinity of this cluster, while two insertion sequence (IS) elements occur in the border and in the middle region of the T3SS-2 gene cluster (Figure 4). The GC content of the T3SS-2 cluster in the P. syringae strains is close to the chromosome average (58–61%), which might
suggest that it has been resident in the P. syringae’s genome for a long time . The codon usage indexes (Additional file 7: Table S2) of the T3SS-2 cluster show the same degree of codon usage bias as the hrc-hrp1 T3SS cluster of P. syringae pv phaseolicola 1448a. Furthermore, the GC content in the third coding position (GC3) of various genes across the T3SS-2 is close to the respective mean of the genome GC3, as in the case of Hrc-Hrp1 (Additional file 7: Table S2). These equal GC levels could indicate an ancient acquisition of the T3SS-2 gene cluster Selleck CP-690550 by P. syringae that was lost in some of its strains. However the scenario of a more recent acquisition from a hypothetical donor with equal GC levels can not be excluded. Evidence for expression of the P. syringae T3SS-2 There are no reports so far for the expression or function of T3SS-2 in members of P. syringae. To obtain preliminary expression evidence of functional putative RNA transcripts,
the hrc II N (sctN) and hrc II C1 (sctC) from P. syringae pv phaseolicola 1448a were detected by RT-PCR in total RNA extracts from cultures grown in rich (LB) ID-8 and minimal (M9) media, after exhaustive treatment with RNase-free DNase I (Supplier Roche Applied Science). Putative transcripts were detected under both growth conditions that were tested, using equal amounts of the extracted total RNA as an RT-PCR template. Interestingly, the detected transcript levels were remarkably higher in LB medium (Figure 3), compared to minimal (M9) medium, probably indicating that the genes are expressed in both cultivation conditions. Conclusions Rhizobia are α-proteobacteria that are able to induce the formation of nodules on leguminous plant roots, where nitrogen fixation takes place with T3SS being one important determinant of this symbiosis [36, 46, 47]. Sequences of the symbiotic plasmids of Rhizobium strains NGR234 and R. etli CFN42 together with the chromosomal symbiotic regions of B. japonicum USDA110 and Mesorhizobium loti R7A have been recently reported [36–38].