Sequence threading techniques and fold-recognition algorithms were used to identify distant homologs. 3-D structural profiles for T3SS proteins were predicted from sequence data was performed using the PHYRE pipeline [16]. The program Memstat3 [17] was used for the prediction of membrane α-helices in proteins. Nucleotide sequence analysis The gene synteny of the T3SS-2 clusters of P. syringae pv phaseolicola 1448a, P. syringae pv oryzae str. 1_6, P. syringae pv tabaci ATCC11528, Rhizobium spp. NGR234 and the gene synteny of the unique T3SS gene clusters of B. japonicum USDA 110, R. etli CIAT 652, R. etli CNF 42, were

compared to other known T3SS gene clusters

of various bacteria using the BLASTN and BLASTP tools of the Genbank. Codon Usage Bias analysis was performed using DnaSP v5 [18]. Phylogenetic analysis T3SS core protein sequences were retrieved using STI571 research buy Psi-BLAST searches with the P. syringae pv phaseolicola 1448a T3SS-2 gene cluster coding frames and were aligned with the multiple alignment method ClustalW, version 1.8 [19]. Phylogenetic relations were inferred using the neighbour-joining method [20] implemented in the MEGA4 software [21]. The bootstrap consensus tree inferred from 1000 replicates [22] is taken to represent the evolutionary history of the amino acid sequences analyzed [22]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates

are collapsed. The percentage of CH5183284 ic50 replicate trees in which the associated taxa Ro 61-8048 solubility dmso clustered together in the bootstrap test (1000 replicates) are shown next to the branches [22]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [23] and are in the units of the number of amino acid substitutions per site. All positions containing alignment gaps and missing data were eliminated only in pair wise Phosphoribosylglycinamide formyltransferase sequence comparisons. Cultivation P. syringae strains were routinely grown at 28°C in LB medium. Bacteria of overnight culture were collected at an OD (optical density) of 0.8. The bacterial pellet was washed with 10 mM MgCl2 and the cells were resuspended (OD: 0.6-0.7) in Hrp-induction media [24] for overnight cultivation at 28°C. The next day the bacterial cells were collected (OD: 0.7-0.8) for RNA extraction. RT-PCR For the RT-PCR reactions, total RNA was extracted from overnight bacterial cultures of P. syringae pv phaseolicola 1448a and P. syringae pv tomato DC3000, using both LB and Hrp-induction media [24]. Total RNA was treated with RNase-free DNase I for 45 min at 37°C [25].