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Involvement of the XpsN protein in formation of the XpsL-xpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus.

ABSTRACT: The xps gene cluster is required for the second step of type II protein secretion in Xanthomonas campestris pv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from the xpsM mutant strain, and vice versa. Introduction of the wild-type xpsM gene carried on a plasmid into the xpsM mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the xpsN mutant strain. They were recovered either by reintroducing the wild-type xpsN gene or by introducing extra copies of wild-type xpsL or xpsM individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of X. campestris pv. campestris caused inhibition of secretion. Complementation of an xpsL or xpsM mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the xpsN gene on the plasmid along with the xpsL and the xpsM genes caused more severe inhibition in both cases. Furthermore, complementation of the xpsN mutant strain was also inhibited. In both the wild-type strain and a strain with the xps gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.

SUBMITTER: Lee HM

PROVIDER: S-EPMC94908 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Involvement of the XpsN protein in formation of the XpsL-xpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus.

Lee H M HM Tyan S W SW Leu W M WM Chen L Y LY Chen D C DC Hu N T NT

Journal of bacteriology 20010101 2

The xps gene cluster is required for the second step of type II protein secretion in Xanthomonas campestris pv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from the xpsM mutant strain, and vice versa. Introducti ...[more]

PMID: 11133946

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Project description:In many plant-bacterial interactions, loss of the type III secretion system (T3SS) severely reduces bacterial growth, symptom causation and suppression of defences in host plants. In the present study of Xanthomonas campestris pv. campestris (Xcc), Xcc strain B305 grew better than strain B186 in Arabidopsis thaliana after hydathode inoculation, and B305 strains mutated to the loss of T3SS (ΔhrcC and/or ΔhrpE; also ΔhrcCΔflgBC) grew similarly to wild-type B305 in Arabidopsis leaves. Unlike Xcc strain B186, wild-type B305 was relatively inefficient in secreting the exogenous T3S effector AvrBsT, but ΔhrcC and/or ΔhrpE attenuated the disease symptoms caused by Xcc B305, showing that the partially compromised T3SS of this strain still promotes necrotic leaf symptoms. In contrast with the T3SS-dependent defence suppression that has been observed for some other plant pathogenic bacteria, the Xcc B186 and B305 wild-type strains (which are virulent on Arabidopsis) caused greater elicitation of host PR-1 and PR-5 expression and callose deposition in comparison with their respective T3SS mutants. A defence-suppressing/virulence-enhancing activity of the Xcc T3SS effector suite was detectable when co-inoculation with wild-type Xcc B186 increased the growth of ΔhrcC Xcc, but this activity did not prevent the above defence elicitation. Experiments using T3SS mutants and Arabidopsis fls2 mutants suggested that FLS2 does not play a prominent role in restriction of the examined Xcc strains. However, ectopic overexpression of the Pseudomonas syringae effector AvrPto promoted in planta growth of wild-type and ΔhrcC Xcc. In summary, the T3SS components or effector suite from virulent Xcc strains elicit some host defence responses, but suppress other defences and stimulate more severe disease symptoms, AvrPto-disruptable elements other than FLS2 apparently contribute to the host restriction of Xcc, and in some virulent Xcc strains the T3SS is not absolutely required for wild-type levels of bacterial growth within the plant.

Project description:Many plant-pathogenic bacteria utilize type II secretion (T2S) systems to secrete degradative enzymes into the extracellular milieu. T2S substrates presumably mediate the degradation of plant cell wall components during the host-pathogen interaction and thus promote bacterial virulence. Previously, the Xps-T2S system from Xanthomonas campestris pv. vesicatoria was shown to contribute to extracellular protease activity and the secretion of a virulence-associated xylanase. The identities and functions of additional T2S substrates from X. campestris pv. vesicatoria, however, are still unknown. In the present study, the analysis of 25 candidate proteins from X. campestris pv. vesicatoria led to the identification of two type II secreted predicted xylanases, a putative protease and a lipase which was previously identified as a virulence factor of X. campestris pv. vesicatoria. Studies with mutant strains revealed that the identified xylanases and the protease contribute to virulence and in planta growth of X. campestris pv. vesicatoria. When analyzed in the related pathogen X. campestris pv. campestris, several T2S substrates from X. campestris pv. vesicatoria were secreted independently of the T2S systems, presumably because of differences in the T2S substrate specificities of the two pathogens. Furthermore, in X. campestris pv. vesicatoria T2S mutants, secretion of T2S substrates was not completely absent, suggesting the contribution of additional transport systems to protein secretion. In line with this hypothesis, T2S substrates were detected in outer membrane vesicles, which were frequently observed for X. campestris pv. vesicatoria. We, therefore, propose that extracellular virulence-associated enzymes from X. campestris pv. vesicatoria are targeted to the Xps-T2S system and to outer membrane vesicles.The virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S substrates are being studied in several plant-pathogenic bacteria, including Xanthomonas campestris pv. vesicatoria, which causes bacterial spot disease in tomato and pepper. Here, we show that the T2S system from X. campestris pv. vesicatoria secretes virulence-associated xylanases, a predicted protease, and a lipase. Secretion assays with the related pathogen X. campestris pv. campestris revealed important differences in the T2S substrate specificities of the two pathogens. Furthermore, electron microscopy showed that T2S substrates from X. campestris pv. vesicatoria are targeted to outer membrane vesicles (OMVs). Our results, therefore, suggest that OMVs provide an alternative transport route for type II secreted extracellular enzymes.

Dataset Information

Involvement of the XpsN protein in formation of the XpsL-xpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus.

Publications

Involvement of the XpsN protein in formation of the XpsL-xpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus.

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