Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Sep 2:12:188.
doi: 10.1186/1471-2180-12-188.

Phylogenetic analysis of a gene cluster encoding an additional, rhizobial-like type III secretion system that is narrowly distributed among Pseudomonas syringae strains

Anastasia D Gazi  1 , Panagiotis F Sarris, Vasiliki E Fadouloglou, Spyridoula N Charova, Nikolaos Mathioudakis, Nicholas J Panopoulos, Michael Kokkinidis
Affiliations

Phylogenetic analysis of a gene cluster encoding an additional, rhizobial-like type III secretion system that is narrowly distributed among Pseudomonas syringae strains

Anastasia D Gazi et al. BMC Microbiol. .

Abstract

Background: The central role of Type III secretion systems (T3SS) in bacteria-plant interactions is well established, yet unexpected findings are being uncovered through bacterial genome sequencing. Some Pseudomonas syringae strains possess an uncharacterized cluster of genes encoding putative components of a second T3SS (T3SS-2) in addition to the well characterized Hrc1 T3SS which is associated with disease lesions in host plants and with the triggering of hypersensitive response in non-host plants. The aim of this study is to perform an in silico analysis of T3SS-2, and to compare it with other known T3SSs.

Results: Based on phylogenetic analysis and gene organization comparisons, the T3SS-2 cluster of the P. syringae pv. phaseolicola strain is grouped with a second T3SS found in the pNGR234b plasmid of Rhizobium sp. These additional T3SS gene clusters define a subgroup within the Rhizobium T3SS family. Although, T3SS-2 is not distributed as widely as the Hrc1 T3SS in P. syringae strains, it was found to be constitutively expressed in P. syringae pv phaseolicola through RT-PCR experiments.

Conclusions: The relatedness of the P. syringae T3SS-2 to a second T3SS from the pNGR234b plasmid of Rhizobium sp., member of subgroup II of the rhizobial T3SS family, indicates common ancestry and/or possible horizontal transfer events between these species. Functional analysis and genome sequencing of more rhizobia and P. syringae pathovars may shed light into why these bacteria maintain a second T3SS gene cluster in their genome.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Evolutionary relationships of SctU proteins. The yellow star indicates the position of the P. syringae pv phaseolicola 1448a HrcIIU. A. The phylogram of 192 SctU sequences with the eight main families named according to Troisfontaines & Cornelis (2005) [8], while the flagellum proteins are depicted in black. The T3SS family encompasing the β-rhizobium Cupriavidus taiwanensis and of Burkholderia cenocepacia group is indicated here with a light purple color (marked as β-Rhc). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. There were a total of 686 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 [21]. B. The Rhc T3SS clade as derived from the phylogram in A, groups the P. syringae HrcIIU sequences close to the RhcIIU protein of the Rhizobium sp. NGR234 T3SS-2. The values at the nodes are the bootstrap percentages out of 1000 replicates. The locus numbers or the protein accession number of each sequence is indicated.
Figure 2
Figure 2
Evolutionary relationships of SctV proteins. Classification of the SctV T3SS proteins into the main T3SS/flagellar families. The colouring scheme of Figure 1 is used.
Figure 3
Figure 3
RT-PCR analysis for the PSPPH_2530, PSPPH_2524 and 16S gene expression in bacterial total RNA. A. RT-PCR analysis for the PSPPH_2524 expression: 1) on total RNA from P. syringae pv phaseolicola 1448a cultivated in Hrp-induction medium, 2) on total RNA from P. syringae pv phaseolicola 1448a cultivated in LB medium, 3) on total RNA from P. syringae pv tomato DC3000 cultivated in LB medium (as a negative control). B. RT-PCR analysis for the PSPPH_2530 expression: 1) on total RNA from P. syringae pv phaseolicola 1448a cultivated in Hrp-induction medium, 2) on total RNA from P. syringae pv phaseolicola 1448a cultivated in LB medium, 3) on total RNA from P. syringae pv tomato DC3000 cultivated in LB medium (as a negative control). C. RT-PCR analysis for the 16S rDNA expression (as a positive control): 1) on total RNA from P. syringae pv phaseolicola 1448a cultivated in Hrp-induction medium, 2) on total RNA from P. syringae pv phaseolicola 1448a cultivated in LB medium, 3) on total RNA from P. syringae pv tomato DC3000 cultivated in LB medium. D. Negative control PCR was performed on the total RNA isolates from 1)P. syringae pv phaseolicola 1448a cultivated in Hrp-induction medium 2)P. syringae pv phaseolicola 1448a cultivated in LB medium, 3)P. syringae pv tomato DC3000 cultivated in LB medium, without Reverse Transcriptase assay using the 16S rDNA primers in order to accredit no DNA contamination in the total RNA samples. PCR products were electrophoretically resolved on ethidium bromide (0.5 μg mL-1)-containing agarose gels (1.5%, w/v). M1: λ DNA digested with PstI, M2: λ DNA digested with EcoRI-HindIII. Even though the total mRNA templates were equal for all PCR samples, the signals in hrp induction medium are very weak, so they have been highlighted by an arrow.
Figure 4
Figure 4
Genetic organization of the Rhc T3SS gene clusters, indicating the diversification of three main subgroups. ORFs are represented by arrows. White arrows indicate either low sequence similarities between syntenic ORFs like the PSPPH_2532: hrpOII case or ORFs not directly related to the T3SS gene clusters that were excluded from the study. Homologous ORFs are indicated by similar coloring or shading pattern. Only a few loci numbers are marked for reference. Gene symbols (N, E, J etc.) for the T3SS-2 genes are following the Hrc1 nomenclature. 1) Subgroup I cluster (Rhc-I), is represented by Bradyrizhobium japonicum USDA110 and includes also the T3SS present on the pNGR234a plasmid of strain NGR234 (not shown); 2) Subgroup II (HrcII/RhcII), represented by the T3SS-II gene clusters of Rhizobium sp. NGR234 pNGR234b plasmid [38] , P. syringae pv phaseolicola 1448A[44], P. syringae pv tabaci ATCC 11528 and P. syringae pv oryzae str. 1_6 (this study, see Materials and Methods); and 3) subgroup III, represented by the sole T3SS of the Rhizobium etli CIAT652 (plasmid b) and the R. etli CNF42 plasmid d [37]. Gene products of the HrcII/RhcII supgroup II T3SS share greater sequence homologies with each other than with genes of subgroups I and III (Additional file 4: Table S1).
Figure 5
Figure 5
Predicted PSPPH_2539 protein domain structure based on fold recognition analysis. See text for details on the various structural templates used. Black dots connect the C-terminus of one threading domain with the N-terminus of the following domain. Residues 195–300 (green segment) are represented separately as an alternative fold for the N-terminal subdomain of the full length AAA+ ATPase domain (yellow).
See this image and copyright information in PMC

References

    1. Economou A, Christie PJ, Fernandez RC, Palmer T, Plano GV, Pugsley AP. Secretion by numbers: protein traffic in prokaryotes. Mol Microbiol. 2006;62(2):308–319. doi: 10.1111/j.1365-2958.2006.05377.x. - DOI - PMC - PubMed
    1. Tseng T-T, Tyler BM, Setubal JC. Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology. BMC Microbiol. 2009;9(Suppl 1):S2. doi: 10.1186/1471-2180-1189-S1181-S1182. - DOI - PMC - PubMed
    1. Cascales E, Christie PJ. The versatile bacterial type IV secretion systems. Nat Rev Microbiol. 2003;1(2):137–149. doi: 10.1038/nrmicro753. - DOI - PMC - PubMed
    1. Cornelis GR. The type III secretion injectisome. Nat Rev Microbiol. 2006;4:811–825. doi: 10.1038/nrmicro1526. - DOI - PubMed
    1. Gazi AD, Charova SN, Panopoulos NJ, Kokkinidis M. Coiled-coils in type III secretion systems: structural flexibility, disorder and biological implications. Cell Microbiol. 2009;11(5):719–729. doi: 10.1111/j.1462-5822.2009.01297.x. - DOI - PubMed

Publication types

Substances

LinkOut - more resources