Abstract
ATP-dependent proteases (FtsH, Lon, and Clp family proteins) are ubiquitous in bacteria and play essential roles in numerous regulatory cell processes. Xanthomonas campestris pv. campestris is a Gram-negative pathogen that can cause black rot diseases in crucifers. The genome of X. campestris pv. campestris has several clp genes, namely, clpS, clpA, clpX, clpP, clpQ, and clpY. Among these genes, only clpX and clpP is known to be required for pathogenicity. Here, we focused on two uncharacterized clp genes (clpS and clpA) that encode the adaptor (ClpS) and ATPase subunit (ClpA) of the ClpAP protease complex. Transcriptional analysis revealed that the expression of clpS and clpA was growth phase-dependent and affected by the growth temperature. The inactivation of clpA, but not of clpS, resulted in susceptibility to high temperature and attenuated virulence in the host plant. The altered phenotypes of the clpA mutant could be complemented in trans. Site-directed mutagenesis revealed that K223 and K504 were the amino acid residues critical for ClpA function in heat tolerance. The protein expression profile shown by the clpA mutant in response to heat stress was different from that exhibited by the wild type. In summary, we characterized two clp genes (clpS and clpA) by examining their expression profiles and functions in different processes, including stress tolerance and pathogenicity. We demonstrated that clpS and clpA were expressed in a temperature-dependent manner and that clpA was required for the survival at high temperature and full virulence of X. campestris pv. campestris. This work represents the first time that clpS and clpA were characterized in Xanthomonas.
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References
Alvarez-Martinez CE et al (2021) Secrete or perish: the role of secretion systems in Xanthomonas biology. Comput Struct Biotechnol J 19:279–302. https://doi.org/10.1016/j.csbj.2020.12.020
An SQ, Tang JL (2018) The Ax21 protein influences virulence and biofilm formation in Stenotrophomonas maltophilia. Arch Microbiol 200:183–187. https://doi.org/10.1007/s00203-017-1433-7
An SQ et al (2020) Mechanistic insights into host adaptation, virulence and epidemiology of the phytopathogen Xanthomonas. FEMS Microbiol Rev 44:1–32. https://doi.org/10.1093/femsre/fuz024
Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. https://doi.org/10.1093/bioinformatics/bti770
Bahar O et al (2014) The Xanthomonas Ax21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles. PeerJ 2:e242. https://doi.org/10.7717/peerj.242
Baker TA, Sauer RT (2006) ATP-dependent proteases of bacteria: recognition logic and operating principles. Trends Biochem Sci 31:647–653. https://doi.org/10.1016/j.tibs.2006.10.006
Belisario JC, Lee HH, Luknauth H, Rigel NW, Martinez LR (2021) Acinetobacter baumannii strains deficient in the Clp chaperone-protease genes have reduced virulence in a murine model of pneumonia. Pathogens. https://doi.org/10.3390/pathogens10020204
Bepperling A et al (2012) Alternative bacterial two-component small heat shock protein systems. Proc Natl Acad Sci U S A 109:20407–20412. https://doi.org/10.1073/pnas.1209565109
Bittner LM, Arends J, Narberhaus F (2016) Mini review: ATP-dependent proteases in bacteria. Biopolymers 105:505–517. https://doi.org/10.1002/bip.22831
Blattner FR et al (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1462. https://doi.org/10.1126/science.277.5331.1453
Brotz-Oesterhelt H, Sass P (2014) Bacterial caseinolytic proteases as novel targets for antibacterial treatment. Int J Med Microbiol 304:23–30. https://doi.org/10.1016/j.ijmm.2013.09.001
Buttner D, Bonas U (2010) Regulation and secretion of Xanthomonas virulence factors. FEMS Microbiol Rev 34:107–133. https://doi.org/10.1111/j.1574-6976.2009.00192.x
Chan JW, Goodwin PH (1999) The molecular genetics of virulence of Xanthomonas campestris. Biotechnol Adv 17:489–508
Chandu D, Nandi D (2004) Comparative genomics and functional roles of the ATP-dependent proteases Lon and Clp during cytosolic protein degradation. Res Microbiol 155:710–719. https://doi.org/10.1016/j.resmic.2004.06.003
Cocotl-Yanez M, Moreno S, Encarnacion S, Lopez-Pliego L, Castaneda M, Espin G (2014) A small heat-shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii, Microbiology (Reading) 160:479–487 doi:https://doi.org/10.1099/mic.0.073353-0
da Silva AC et al (2002) Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature 417:459–463
Denance N, Lahaye T, Noel LD (2016) Editorial: genomics and effectomics of the crop killer Xanthomonas. Front Plant Sci 7:71. https://doi.org/10.3389/fpls.2016.00071
Dougan DA, Reid BG, Horwich AL, Bukau B (2002) ClpS, a substrate modulator of the ClpAP machine. Mol Cell 9:673–683. https://doi.org/10.1016/s1097-2765(02)00485-9
Dow JM, Crossman L, Findlay K, He YQ, Feng JX, Tang JL (2003) Biofilm dispersal in Xanthomonas campestris is controlled by cell-cell signaling and is required for full virulence to plants. Proc Natl Acad Sci USA 100:10995–11000
Ekaza E, Guilloteau L, Teyssier J, Liautard JP, Kohler S (2000) Functional analysis of the ClpATPase ClpA of Brucella suis, and persistence of a knockout mutant in BALB/c mice Microbiology (Reading) 146 ( Pt 7):1605–1616 . https://doi.org/10.1099/00221287-146-7-1605
Erbse A et al (2006) ClpS is an essential component of the N-end rule pathway in E. coli. Nature 439:753–756. https://doi.org/10.1038/nature04412
Erbse AH et al (2008) Conserved residues in the N-domain of the AAA+ chaperone ClpA regulate substrate recognition and unfolding. FEBS J 275:1400–1410. https://doi.org/10.1111/j.1742-4658.2008.06304.x
Feinbaum RL, Urbach JM, Liberati NT, Djonovic S, Adonizio A, Carvunis AR, Ausubel FM (2012) Genome-wide identification of Pseudomonas aeruginosa virulence-related genes using a Caenorhabditis elegans infection model. PLoS Pathog 8:e1002813. https://doi.org/10.1371/journal.ppat.1002813
Fu JF, Tseng YH (1990) Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey. Appl Environ Microbiol 56:919–923
Gottesman S, Clark WP, Maurizi MR (1990) The ATP-dependent Clp protease of E. coli Sequence of clpA and identification of a Clp-Specific substrate. J Biol Chem 265:7886–7893
Guo F, Esser L, Singh SK, Maurizi MR, Xia D (2002) Crystal structure of the heterodimeric complex of the adaptor, ClpS, with the N-domain of the AAA+ chaperone. ClpA J Biol Chem 277:46753–46762. https://doi.org/10.1074/jbc.M208104200
Haslbeck M, Vierling E (2015) A first line of stress defense: small heat shock proteins and their function in protein homeostasis. J Mol Biol 427:1537–1548. https://doi.org/10.1016/j.jmb.2015.02.002
Hoskins JR, Sharma S, Sathyanarayana BK, Wickner S (2001) Clp ATPases and their role in protein unfolding and degradation. Adv Protein Chem 59:413–429. https://doi.org/10.1016/s0065-3233(01)59013-0
Hsiao YM, Liao HY, Lee MC, Yang TC, Tseng YH (2005) Clp upregulates transcription of engA gene encoding a virulence factor in Xanthomonas campestris by direct binding to the upstream tandem Clp sites. FEBS Lett 579:3525–3533
Hsiao YM, Liu YF, Fang MC, Song WL (2011) XCC2731, a GGDEF domain protein in Xanthomonas campestris, is involved in bacterial attachment and is positively regulated by Clp, Microbiol Res 166:548–565 doi:S0944–5013(10)00109–6 [pii]. https://doi.org/10.1016/j.micres.2010.11.003
Katayama Y, Gottesman S, Pumphrey J, Rudikoff S, Clark WP, Maurizi MR (1988) The two-component, ATP-dependent Clp protease of E. coli purification, cloning, and mutational analysis of the ATP-binding component. J Biol Chem 263:15226–15236
Keen NT, Tamaki S, Kobayashi D, Trollinger D (1988) Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70:191–197
Klein G, Laskowska E, Taylor A, Lipinska B (2001) IbpA/B small heat-shock protein of marine bacterium Vibrio harveyi binds to proteins aggregated in a cell during heat shock. Mar Biotechnol (NY) 3:346–354. https://doi.org/10.1007/s10126001-0009-2
Koo BM, Rhodius VA, Campbell EA, Gross CA (2009) Dissection of recognition determinants of E. coli sigma32 suggests a composite -10 region with an “extended -10” motif and a core -10 element. Mol Microbiol 72:815–829. https://doi.org/10.1111/j.1365-2958.2009.06690.x
Kress W, Maglica Z, Weber-Ban E (2009a) Clp chaperone-proteases: structure and function. Res Microbiol 160:618–628. https://doi.org/10.1016/j.resmic.2009.08.006
Kress W, Mutschler H, Weber-Ban E (2009b) Both ATPase domains of ClpA are critical for processing of stable protein structures. J Biol Chem 284:31441–31452. https://doi.org/10.1074/jbc.M109.022319
Kthiri F et al (2010) Protein aggregation in a mutant deficient in YajL, the bacterial homolog of the Parkinsonism-associated protein DJ-1. J Biol Chem 285:10328–10336. https://doi.org/10.1074/jbc.M109.077529
Kumar Verma R, Samal B, Chatterjee S (2018) Xanthomonas oryzae pv. oryzae chemotaxis components and chemoreceptor Mcp2 are involved in the sensing of constituents of xylem sap and contribute to the regulation of virulence-associated functions and entry into rice. Mol Plant Pathol 19:2397–2415. https://doi.org/10.1111/mpp.12718
Laskowska E, Wawrzynow A, Taylor A (1996) IbpA and IbpB, the new heat-shock proteins, bind to endogenous E. coli proteins aggregated intracellularly by heat shock. Biochimie 78:117–122. https://doi.org/10.1016/0300-9084(96)82643-5
Lee TC, Chen ST, Lee MC, Chang CM, Chen CH, Weng SF, Tseng YH (2001) The early stages of filamentous phage phiLf infection require the host transcription factor. Clp J Mol Microbiol Biotechnol 3:471–481
Lee S, Owen HA, Prochaska DJ, Barnum SR (2000) HSP16.6 is involved in the development of thermotolerance and thylakoid stability in the unicellular cyanobacterium, Synechocystis sp. PCC 6803. Curr Microbiol 40:283–287. https://doi.org/10.1007/s002849910056
Lee SW, Han SW, Bartley LE, Ronald PC (2006) From the academy: colloquium review. Unique characteristics of Xanthomonas oryzae pv. oryzae AvrXa21 and implications for plant innate immunity. Proc Natl Acad Sci USA 103:18395–18400. https://doi.org/10.1073/pnas.0605508103
Lee KJ, Jung YC, Park SJ, Lee KH (2018) Role of heat shock proteases in quorum-sensing-mediated regulation of biofilm formation by Vibrio Species, mBio. https://doi.org/10.1128/mBio.02086-17
Li CE, Liao CT, Lo HH, Hsiao YM (2020) Functional characterization and transcriptional analysis of clpP of Xanthomonas campestris pv. campestris. Curr Microbiol 77:2876–2885. https://doi.org/10.1007/s00284-020-02093-1
Lin YM et al (2008) Transposon mutagenesis reveals differential pathogenesis of Ralstonia solanacearum on tomato and Arabidopsis. Mol Plant Microbe Interact 21:1261–1270. https://doi.org/10.1094/MPMI-21-9-1261
Lin CH, Lee CN, Lin JW, Tsai WJ, Wang SW, Weng SF, Tseng YH (2010) Characterization of Xanthomonas campestris pv. campestris heat shock protein A (HspA), which possesses an intrinsic ability to reactivate inactivated proteins. Appl Microbiol Biotechnol 88:699–709. https://doi.org/10.1007/s00253-010-2776-z
Liu YC et al (2015) Complete genome sequence of Xanthomonas campestris pv. campestris strain 17 from Taiwan. Genome Announc. https://doi.org/10.1128/genomeA.01466-15
Lo HH, Liao CT, Li CE, Chiang YC, Hsiao YM (2020) The clpX gene plays an important role in bacterial attachment, stress tolerance, and virulence in Xanthomonas campestris pv. campestris. Arch Microbiol 202:597–607. https://doi.org/10.1007/s00203-019-01772-3
Mahmoud SA, Chien P (2018) Regulated proteolysis in bacteria. Annu Rev Biochem 87:677–696. https://doi.org/10.1146/annurev-biochem-062917-012848
Malik IT, Brotz-Oesterhelt H (2017) Conformational control of the bacterial Clp protease by natural product antibiotics. Nat Prod Rep 34:815–831. https://doi.org/10.1039/c6np00125d
Miller JM, Enemark EJ (2016) Fundamental characteristics of AAA+ protein family structure and function. Archaea 2016:9294307. https://doi.org/10.1155/2016/9294307
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
Mogk A, Deuerling E, Vorderwulbecke S, Vierling E, Bukau B (2003a) Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation. Mol Microbiol 50:585–595. https://doi.org/10.1046/j.1365-2958.2003.03710.x
Mogk A, Schlieker C, Friedrich KL, Schonfeld HJ, Vierling E, Bukau B (2003b) Refolding of substrates bound to small Hsps relies on a disaggregation reaction mediated most efficiently by ClpB/DnaK. J Biol Chem 278:31033–31042. https://doi.org/10.1074/jbc.M303587200
Mogk A, Ruger-Herreros C, Bukau B (2019) Cellular functions and mechanisms of action of small heat shock proteins. Annu Rev Microbiol 73:89–110. https://doi.org/10.1146/annurev-micro-020518-115515
Nonaka G, Blankschien M, Herman C, Gross CA, Rhodius VA (2006) Regulon and promoter analysis of the E. coli heat-shock factor, sigma32, reveals a multifaceted cellular response to heat stress. Genes Dev 20:1776–1789. https://doi.org/10.1101/gad.1428206
Obuchowski I, Karas P, Liberek K (2021) The small ones matter-sHsps in the bacterial chaperone network. Front Mol Biosci 8:666893. https://doi.org/10.3389/fmolb.2021.666893
Olivares AO, Baker TA, Sauer RT (2018) Mechanical protein unfolding and degradation. Annu Rev Physiol 80:413–429. https://doi.org/10.1146/annurev-physiol-021317-121303
Pak M, Wickner S (1997) Mechanism of protein remodeling by ClpA chaperone. Proc Natl Acad Sci U S A 94:4901–4906. https://doi.org/10.1073/pnas.94.10.4901
Pak M, Hoskins JR, Singh SK, Maurizi MR, Wickner S (1999) Concurrent chaperone and protease activities of ClpAP and the requirement for the N-terminal ClpA ATP binding site for chaperone activity. J Biol Chem 274:19316–19322. https://doi.org/10.1074/jbc.274.27.19316
Park HJ, Lee SW, Han SW (2014) Proteomic and functional analyses of a novel porin-like protein in Xanthomonas oryzae pv. oryzae. J Microbiol 52:1030–1035. https://doi.org/10.1007/s12275-014-4442-0
Qian W et al (2005) Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris. Genome Res 15:757–767
Qian G et al (2013) Proteomic analysis reveals novel extracellular virulence-associated proteins and functions regulated by the diffusible signal factor (DSF) in Xanthomonas oryzae pv. oryzicola. J Proteome Res 12:3327–3341. https://doi.org/10.1021/pr4001543
Ratajczak E, Zietkiewicz S, Liberek K (2009) Distinct activities of E. coli small heat shock proteins IbpA and IbpB promote efficient protein disaggregation. J Mol Biol 386:178–189. https://doi.org/10.1016/j.jmb.2008.12.009
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd. Cold Spring Harbor Press, Cold Spring Harbor, N. Y.
Sangpuii L et al (2018) Comparative roles of clpA and clpB in the survival of S. Typhimurium under stress and virulence in poultry. Sci Rep 8:4481. https://doi.org/10.1038/s41598-018-22670-6
Sauer RT, Baker TA (2011) AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev Biochem 80:587–612. https://doi.org/10.1146/annurev-biochem-060408-172623
Schmidt R, Zahn R, Bukau B, Mogk A (2009) ClpS is the recognition component for E. coli substrates of the N-end rule degradation pathway. Mol Microbiol 72:506–517. https://doi.org/10.1111/j.1365-2958.2009.06666.x
Schweizer HD (1993) Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. Biotechniques 15:831–834
Sen A, Zhou Y, Imlay JA (2020) During oxidative stress the Clp proteins of E. coli ensure that iron pools remain sufficient to reactivate oxidized metalloenzyme. J Bacteriol. https://doi.org/10.1128/JB.00235-20
Seol JH, Baek SH, Kang MS, Ha DB, Chung CH (1995) Distinctive roles of the two ATP-binding sites in ClpA, the ATPase component of protease Ti in Escherichia coli. J Biol Chem 270:8087–8092. https://doi.org/10.1074/jbc.270.14.8087
da Silva FG, Shen Y, Dardick C, Burdman S, Yadav RC, de Leon AL, Ronald PC (2004) Bacterial genes involved in type I secretion and sulfation are required to elicit the rice Xa21-mediated innate immune response. Mol Plant Microbe Interact 17:593–601. https://doi.org/10.1094/MPMI.2004.17.6.593
Singh SK, Maurizi MR (1994) Mutational analysis demonstrates different functional roles for the two ATP-binding sites in ClpAP protease from Escherichia coli. J Biol Chem 269:29537–29545
Suzuki CK, Rep M, van Dijl JM, Suda K, Grivell LA, Schatz G (1997) ATP-dependent proteases that also chaperone protein biogenesis. Trends Biochem Sci 22:118–123. https://doi.org/10.1016/s0968-0004(97)01020-7
Tang JL, Tang DJ, Dubrow ZE, Bogdanove A, An SQ (2021) Xanthomonas campestris pathovars. Trends Microbiol 29:182–183. https://doi.org/10.1016/j.tim.2020.06.003
Thomas JG, Baneyx F (1998) Roles of the Escherichia coli small heat shock proteins IbpA and IbpB in thermal stress management: comparison with ClpA. ClpB, and HtpG in Vivo J Bacteriol 180:5165–5172. https://doi.org/10.1128/JB.180.19.5165-5172.1998
Torres-Delgado A, Kotamarthi HC, Sauer RT, Baker TA (2020) The intrinsically disordered N-terminal extension of the ClpS adaptor reprograms its partner AAA+ ClpAP protease. J Mol Biol 432:4908–4921. https://doi.org/10.1016/j.jmb.2020.07.007
Vieira J, Messing J (1991) New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100:189–194
Vorholter FJ et al (2008) The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis. J Biotechnol 134:33–45
Wang TW, Tseng YH (1992) Electrotransformation of Xanthomonas campestris by RF DNA of filamentous phage ϕLf. Lett Appl Microbiol 14:65–68
Wawrzynow A, Banecki B, Zylicz M (1996) The Clp ATPases define a novel class of molecular chaperones. Mol Microbiol 21:895–899. https://doi.org/10.1046/j.1365-2958.1996.421404.x
Wendler P, Ciniawsky S, Kock M, Kube S (2012) Structure and function of the AAA+ nucleotide binding pocket. Biochim Biophys Acta 1823:2–14. https://doi.org/10.1016/j.bbamcr.2011.06.014
Wickner S, Gottesman S, Skowyra D, Hoskins J, McKenney K, Maurizi MR (1994) A molecular chaperone, ClpA, functions like DnaK and DnaJ. Proc Natl Acad Sci USA 91:12218–12222. https://doi.org/10.1073/pnas.91.25.12218
Xia D, Esser L, Singh SK, Guo F, Maurizi MR (2004) Crystallographic investigation of peptide binding sites in the N-domain of the ClpA chaperone. J Struct Biol 146:166–179. https://doi.org/10.1016/j.jsb.2003.11.025
Yang BY, Tseng YH (1988) Production of exopolysaccharide and levels of protease and pectinase activity in pathogenic and non-pathogenic strains of Xanthomonas campestris pv. Campestris. Bot Bull Acad Sin 29:93–99
Yeom J, Gao X, Groisman EA (2018) Reduction in adaptor amounts establishes degradation hierarchy among protease substrates. Proc Natl Acad Sci U S A 115:E4483–E4492. https://doi.org/10.1073/pnas.1722246115
Zeth K, Ravelli RB, Paal K, Cusack S, Bukau B, Dougan DA (2002) Structural analysis of the adaptor protein ClpS in complex with the N-terminal domain of ClpA. Nat Struct Biol 9:906–911. https://doi.org/10.1038/nsb869
Zgurskaya HI, Krishnamoorthy G, Ntreh A, Lu S (2011) Mechanism and function of the outer membrane channel TolC in multidrug resistance and physiology of enterobacteria. Front Microbiol 2:189. https://doi.org/10.3389/fmicb.2011.00189
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This study was supported by the Ministry of Science and Technology of Taiwan (Grants No. MOST 107-2313-B-166-001-MY3 and MOST 110-2313-B-166-001).
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Lo, HH., Chang, HC., Liao, CT. et al. Expression and function of clpS and clpA in Xanthomonas campestris pv. campestris. Antonie van Leeuwenhoek 115, 589–607 (2022). https://doi.org/10.1007/s10482-022-01725-9
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DOI: https://doi.org/10.1007/s10482-022-01725-9