Expression and Function of clpS and clpA in Xanthomonas Campestris Pv. Campestris

doi:10.21203/rs.3.rs-774551/v1

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Expression and Function of clpS and clpA in Xanthomonas Campestris Pv. Campestris

https://doi.org/10.21203/rs.3.rs-774551/v1

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published 23 Mar, 2022

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ATP-dependent proteases (FtsH, Lon, and Clp family proteins) are ubiquitous in bacteria and play essential roles in many regulatory cell processes. Xanthomonas campestris pv. campestris is a Gram-negative pathogen and can cause black rot diseases in crucifers. The genome of X. campestris pv. campestris have several clp genes, namely clpS, clpA, clpX, clpP, clpQ, and clpY. Among them, only clpX and clpP were 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, respectively. Transcriptional analysis revealed that expression of both clpS and clpA is induced by heat shock. Inactivation of clpA but not clpS resulted in susceptibility to high temperature and revealed an attenuation of virulence on the host plant. The altered phenotypes of the clpA mutant could be complemented in trans. Site-directed mutagenesis revealed that K223 and K504 are critical amino acid residues for ClpA function in heat tolerance. The clpA mutant revealed different protein expression profiles as compared to the wild type in response to heat stress. Taken together, we characterized two clp genes (clpS and clpA) by examining their expression and function including stress tolerance and pathogenicity. We demonstrated that both clpS and clpA are expressed in a temperature dependent manner, and clpA is required for survival at high temperature and full virulence of X. campestris pv. campestris. This is the first time that clpS and clpA has been characterized in Xanthomonas.

Molecular Biology

General Microbiology

pathogenicity

stress tolerance

transcription

Xanthomonas

Regulated proteolysis is an essential process that affects several biological pathways (Mahmoud and Chien 2018). In bacteria, regulated proteolysis is carried out by energy-dependent AAA+ (ATPases associated with cellular activities) proteases that use the power of ATP hydrolysis to recognize, unfold, translocate, and degrade substrates (Mahmoud and Chien 2018). AAA+ proteases are not only required for regulated proteolysis but also essential for quality control of aberrant or aggregated proteins under certain conditions (Bittner et al. 2016).

Several AAA+ proteases exist in bacteria, such as FtsH, Lon, and Clp family proteases [ClpAP, ClpXP, and ClpYQ (HslUV)] (Bittner et al. 2016, Mahmoud and Chien 2018, Sauer and Baker 2011). All these AAA+ proteases are compartmentalized proteases that consist of two distinct functional units with separate activities: ATPase and protease (Baker and Sauer 2006, Bittner et al. 2016, Mahmoud and Chien 2018). In the case of FtsH and Lon, the ATPase domain and the protease domain are located on a single polypeptide (Bittner et al. 2016, Mahmoud and Chien 2018, Sauer and Baker 2011). In the Clp family, the AAA+ family ATPase subunit [ClpA, ClpX, and ClpY (HslU)] and the proteolytic subunit [ClpP and ClpQ (HslV)] are encoded in distinct polypeptide chains (Bittner et al. 2016, Mahmoud and Chien 2018, Sauer and Baker 2011). ClpA and ClpX function as chaperones and bind to ClpP (serine protease) to form the ATP-dependent proteases ClpAP or ClpXP, whereas ClpY (HslU) interacts with ClpQ (HslV) to form the ClpYQ (HslUV) protease (Chandu and Nandi 2004).

In the ClpAP protease, one or two AAA+ ClpA hexamers associated with the tetradodecameric ClpP to perform ATP-dependent protein degradation (Olivares et al. 2018). An adaptor protein may also be associated, such as ClpS, which interacts with the N-domain of ClpA and influences the ClpAP complex (Erbse et al. 2006). ClpAP exists in most Gram-negative proteobacteria and is characterized extensively in the model organism Escherichia coli (Kress et al. 2009a). ClpS is a highly conserved protein and ClpS homologs are found not only in bacterial but also in plant species (Dougan et al. 2002). As part of ClpAP complex, ClpA has several functions: ATP hydrolysis, substrate recognition, protein unfolding as well as translocation to its proteolytic partner ClpP (Xia et al. 2004). In the absence of the proteolytic component ClpP, ClpA functions as a molecular chaperone and catalyzes protein unfolding and limited protein remodeling (Wickner et al. 1994, Xia et al. 2004). ClpS is able to regulate ClpA substrate selection and is an essential component of the N-end rule pathway (Erbse et al. 2006).

Xanthomonas campestris pv. campestris is an important plant pathogen that causes a systemic vascular disease of crucifers known as black rot (An et al. 2020). Six predicted Clp family protein-encoding genes were annotated in the genome of X. campestris pv. campestris (da Silva et al. 2002, Liu et al. 2015, Qian et al. 2005, Vorholter et al. 2008). They are adaptor protein encoding gene (clpS), genes coding for AAA+ ATPase (clpA, clpX, and clpY), and genes coding for Clp protease (clpP, and clpQ). Among them, only two (clpX and clpP) have been studied. The X. campestris pv. campestris clpX was found to play an important role in bacterial attachment, stress tolerance, and virulence (Lo et al. 2020). The X. campestris pv. campestris clpP was showed to be required for growth under heat stress and in the presence of puromycin, essential for full virulence, and its expression was induced by heat shock (Li et al. 2020). In this study, we aimed to characterize clpS and clpA in X. campestris pv. campestris. We employed transcriptional fusion assay to evaluate the expression of clpS and clpA. In addition, we utilized deletion mutants of clpS and clpA, along with their genetic complements, to investigate the functions of the corresponding proteins in X. campestris pv. campestris stress tolerance and pathogenicity.

Strains, media, and culture conditions

The E. coli ECOS^TM 101 Competent Cells [DH5a] used as the host for DNA cloning were purchased from Yeastern Biotech. The X. campestris pv. campestris wild-type strain Xcc17 was isolated in Taiwan (Yang and Tseng 1988). The Xcc17-derived mutant strains obtained by marker exchange included DclpS (clpS mutant) and DclpA (clpA mutant) were constructed in the present study. All bacteria cultures were routinely cultured in Luria–Bertani (LB) medium (Miller 1972), unless otherwise noted, and were grown at incubated 37 ºC for E. coli and 28 ºC for X. campestris pv. campestris strains, respectively. XOLN supplemented with glycerol (2%) was a basal salt medium (Fu and Tseng 1990). Ampicillin (50 μg/mL), kanamycin (50 μg/mL), gentamycin (15 μg/mL), and tetracycline (15 μg/mL) were added to the medium as required. For liquid cultures, bacteria cells were grown with shaking at 180 revolutions per minute (rpm). The agar concentration used for solid media was 1.5%.

Molecular techniques

The restriction enzymes, T4 DNA ligase, and Taq DNA polymerase used in this study were purchased from Promega, Roche, and Yeastern. DNA manipulation techniques and DNA transformation of E. coli were performed using standard methods as described (Sambrook et al. 1989). Genomic DNA and plasmid DNA were prepared using the Wizard^ÒGenomic DNA Purification Kit (Promega) and the Gene-Spin™ Miniprep Purification Kit (Protech), respectively. The polymerase chain reaction (PCR) was performed as previously described (Hsiao et al. 2005) using the primers listed in Table 1. DNA sequences were determined by Mission Biotech Co., Ltd. (Taipei, Taiwan). Transformation of X. campestris pv. campestris was carried out by electroporation (Wang and Tseng 1992).

Upstream region construction and promoter activity assay

DNA fragments containing the upstream regions of clpS and clpA were PCR-amplified using primer pair 35PstI/476XbaI and 621PstI/946XbaI, ligated into cloning vector yT&A (Yeastern), generating pTclpS and pTclpA. After sequence confirmation, the insert in pTclpS and pTclpA was excised and cloned ahead of a promoterless lacZ gene (reporter) in pFY13–9 (Lee et al. 2001), giving pFYclpS and pFYclpA. The resulting plasmids carried nt –448/–7 (442 bp) and –326/–1 (326 bp) region relative to the clpS and clpA putative translational start point, respectively (Fig. 1).

For promoter activity assay, the X. campestris pv. campestris wild-type Xcc17 carrying these constructs were cultured overnight and inoculated into fresh media to obtain an initial optical density at 550 nm (OD₅₅₀) of 0.35. Then, the cultures were incubated either at 28 °C or at 37 °C. Aliquots were withdrawn at 6, 24, 30 and 48 h post-incubation and the b-galactosidase activity was determined as described previously (Miller 1972). The experiment was performed in duplicate and repeated at least three times. Data represent the average of three replicates.

Sequence analysis and homology modeling

The nucleotide and the predicted amino acid sequence as well as the sequence comparison were analyzed online (http://www.ncbi.nlm.nih.gov). The three-dimensional structural model of Xcc17 ClpS and ClpA was based on the E. coli strain K-12 ClpS (PDB ID 2W9R) and ClpA (PDB ID 1KSF) from RCSB PDB database. They shared 56.4% and 63.0% sequence identity with Xcc17 ClpS and ClpA, respectively. The homology modeling structure of Xcc17 ClpS and ClpA used the project mode with the SWISS-MODEL workspace (https://swissmodel.expasy.org) (Arnold et al. 2006). The web-based application of NGL viewer (http://proteinformatics.charite.de/ngl) was used for the molecular visualization of protein structure.

Mutant construction and complementation

To construct the clpS mutant, the 135 bp PstI-EcoRI fragment in pTclpA was excised and cloned into pOK12 (Vieira and Messing 1991). The obtained plasmid pOKclpS, containing the internal region of the Xcc17 clpS gene, was then transferred into Xcc17 by electroporation following a single crossover. For the clpA mutant construction, the 2334-bp HindIII-XbaI fragment encompassing the upstream 48 bp fragment plus the entire coding region of the Xcc17 clpA was synthesized and cloned in pUC57-Mini (Protech), giving pUCclpA. Then, the 693-bp EcoRV-PstI fragment of pUCclpA was excised and cloned into pOK12, giving pOKclpA. A Gm^R cartridge from pUCGM (Schweizer 1993) was inserted into the HincII site within the pOKclpA insert. The resultant plasmid, pOKclpAG, was further transferred into Xcc17 by electroporation allowing for double crossover. Insertion of pOKclpS or Gm^R cartridge into the target gene (clpS or clpA) was verified by PCR. The confirmed clpS and clpA mutant strains were designated as DclpS and DclpA.

For construction of the DclpS complementation plasmid, the 546 bp DNA fragment encompassing the upstream 82 bp fragment plus the entire coding region of the Xcc17 clpS was PCR amplified using primer pair 402HindIII/946XbaI and cloned into the broad-host-range vector pRK415 (Keen et al. 1988), giving pRKclpS (Fig. 1). For construction of the DclpA complementation plasmid, the 2334-bp HindIII-XbaI fragment of pUCclpA was excised and cloned in pRK415, yielding pRKclpA (Fig. 1). For complementation of the clpS and clpA mutants, plasmid pRKclpS and pRKclpA were electroporated into DclpS and DclpA , respectively. The complemented strains were designated as DclpS(pRKclpS) and DclpA(pRKclpA). In parallel, empty vector pRK415 was introduced into Xcc17, DclpS and DclpA, generating Xcc17(pRK415), DclpS(pRK415) and DclpA(pRK415) for comparison.

Site-directed mutagenesis

The site-directed mutagenesis of ClpA was carried out by the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies), according to the manufacturer’s instructions. The mutation was constructed in the most commonly mutated residue in the Walker A motif of AAA⁺ protein (Wendler et al. 2012). The invariant lysine at position 223 and 504 was substituted to glutamine, using pUCclpA as a template for PCR and the primers listed in Table 1. After DNA sequence verification, the mutated clpA was cloned into pRK415 to give pRKclpAK223Q and pRKclpAK504Q (Fig. 1). These resulting constructs were separately introduced into DclpA, yielding DclpA(pRKclpAK223Q) and DclpA(pRKclpAK504Q).

Temperature tolerance assay

The method of temperature tolerance assay was as described earlier (Lo et al. 2020). Briefly, overnight cultures of tested strains were diluted with fresh LB broth to an initial density of OD₅₅₀ = 0.5 (5 × 10⁸ cells/mL) and followed tenfold serial dilutions (5 × 10⁷ to 5 × 10⁵ cells/mL). Then, cell suspensions (5 mL) from each dilution were spotted on LB plates and incubated either at 28 °C or at 37 °C for 4 days. The assay was repeated at least three times, independently, in two replicates for each time.

Cell extracts preparation and analysis

Overnight grown cultures of tested strains were inoculated into fresh XOLN-glycerol medium to obtain an initial OD₅₅₀ = 0.35 and incubated at either at 28 °C or at 37 °C for 24 h. Bacteria cells were collected through centrifugation at 12,000 ´ g for 2 min at 4 °C. Each preparation was obtained from the same number of cells. The cell pellets were rinsed and suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Cell extracts was obtained through sonication (cycles of 4 sec pulse and 5 sec rest on ice for 2 min). Following sonication, the cell debris and unbroken cells were removed by centrifugation at 2,000 ´ g for 2 min at 4 °C, and the supernatant fraction was referred to cell extracts.

Protein aggregates were isolated according to a procedure described previously (Kthiri et al. 2010) with some modifications. In brief, cell extracts were incubated with or without 0.5% Triton X-100 for 15 min at 25 °C. The insoluble cell fractions were recovered by subsequent centrifugation at 8,000 × g for 15 min at 25 °C. The insoluble cell fractions treated with Triton X-100 were referred to protein aggregates, and which omitting Triton X-100 treatment was obtained for comparison. The insoluble cell fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands were stained with Coomassie brilliant blue. The amount of loading was normalized in terms of colony forming units (CFUs) and was equivalent to 4.2 × 10⁸ CFUs/lane.

Virulence assay

The virulence of X. campestris pv. campestris strains on cabbage was determined using the leaf clip inoculation method (Hsiao et al. 2011). Leaves were cut with scissors dipped in the bacterial suspension with cell concentration adjusted to OD₅₅₀ = 1. Lesion lengths were measured and images were taken 14 days post-inoculation. Three independent experiments with six replicates were performed. The values are presented as mean lesion length ± standard deviations.

Expression of clpS and clpA is induced by heat treatment

The genome of the sequenced X. campestris pv. campestris strain 17 (Liu et al. 2015) possesses clpS (locus_tag AAW18_RS09760) and clpA (locus_tag AAW18_RS09765), which adjacent to each other on the chromosome (Fig. 1). Genes infA and aat, coding for translation initiation factor IF-1 and leucyl/phenylalanyl-tRNA-protein transferase, are located downstream of clpA. As shown in Fig. 1, there are such compatible homologs with 49-69% shared amino acid sequence identities in X. campestris pv. campestris strain 17 and E. coli strain K-12 (Blattner et al. 1997). The mutT gene (locus_tag AAW18_RS0975 in X. campestris pv. campestris strain 17), coding for NUDIX hydrolase, is located upstream of clpS, and its homologue appears elsewhere in the chromosome in E. coli K-12 (Blattner et al. 1997). In addition to X. campestris pv. campestris strain 17, the organization of clpS and clpA as well as their flanking genes is similar in several X. campestris pv. campestris strains, such as ATCC33913, 8004, and B100 (da Silva et al. 2002, Qian et al. 2005, Vorholter et al. 2008).

Gene organization analysis showed that clpS and its upstream gene mutT as well as clpA and its downstream gene infA are in the opposite direction, and the clpS and clpA genes are linked together in the same orientation with 143 bp intergenic space (Fig. 1). Owing to its orientation and intergenic regions with flanking genes, clpS and clpA are likely to possessing their own promoter. Sequence analysis revealed that the upstream regions of clpS and clpA shown to resemble the E. coli s³²-dependent heat shock promoter (TTGAAA-N_13-14-CCCCATNT) (Koo et al. 2009, Nonaka et al. 2006). A possible s³² promoter with a –35 box (CGTAAA) and a –10 box (GGCCAAAT) were located at –61 and –39 (with a spacer of 14 nucleotides) relative to the clpS translation start site. A putative s³²-type promoter with –35 box (TGGAAA) and a –10 box (CCGCATAT) separated by 13 nucleotides was found 29 nucleotides upstream of the clpA initiation codon. The presence of a s³²-type promoter led us to predict that the expression of clpS and clpA may be influenced under heat shock condition.

To evaluate the expression of clpS and clpA, the upstream regions of clpS and clpA were cloned ahead of a promoterless lacZ gene in pFY13–9 (Lee et al. 2001) as described in the Materials and methods, yielding pFYclpS and pFYclpS. Then, the generated reporter constructs were introduced into Xcc17 and the resulting strains, Xcc17(pFYclpS) and Xcc17(pFYclpA), were used to evaluate the expression of clpS and clpA. The promoter activities of clpS and clpA were assayed by measuring the b-galactosidase activities in Xcc17(pFYclpS) (Fig. 2a) and Xcc17(pFYclpA) (Fig. 2b) grown at 28 ºC or 37 ºC. When strains grown at normal physiological temperature (28 ºC), the activities measured in Xcc17(pFYclpS) and Xcc17(pFYclpA) were 69 U and 121 U at 6 h, increased following cell growth, and gave highest level at 48 h (343 U and 769 U). When strains grown at 37 ºC (heat stress condition), the b-galactosidase activities levels increased gradually and reached their maxima at 48 h, which were 413 U in Xcc17(pFYclpS) and 1220 U in Xcc17(pFyclpA), respectively, exhibiting approximately 1.6-fold increment in both cases compared with normal growing temperature. Results from reporter assays indicated that the expression of clpS and clpA is growth phase dependent and subjected to heat shock induction. Limited information was available regarding the expression of clpS and clpA, and the effects of heat stress on their expression is not similar among bacteria. The cellular level of ClpA in Vibrio vulnificus was induced under heat shock condition (Lee et al. 2018). In E. coli, the clpA gene has been reported as a monocistronic message whereas the synthesis of ClpA does not increase on heat shock (Gottesman et al. 1990, Katayama et al. 1988). In the case of Brucella suis, the expression of clpA is not increased at high temperature (Ekaza et al. 2000). It is indicated that PhoP represses the transcription of clpS but not clpA in E. coli and Salmonella enterica when bacteria experience a low cytoplasmic Mg²⁺ concentration (Yeom et al. 2018). Recently, it is reported that clpS and clpA is induced during H₂O₂ stress and OxyR is required for H₂O₂-driven induction of clpSA in E. coli (Sen et al. 2020). The effects of OxyR and PhoP on the expression of clpS and clpA in X. campestris pv. campestris remains to be elucidated.

Bioinformatics analyses indicate that ClpS and ClpA retain key residues required for their functionality

The clpS open reading frame is 321 bp in length and located in the genome sequence at position 2227483–2227803 in the Xcc17 chromosome (GenBank accession number NZ_CP011946). The predicted protein encoded by clpS, which consists of 106 amino acids and is annotated as an ATP-dependent Clp protease adaptor ClpS, has a calculated molecular mass of 12601 Da and a pI of 6.03. Domain organization analysis indicated that it has a ClpS domain situated at residues 23–102 (bit score: 113.20, E-value: 1.1e-29) (Fig. 3a). A BLAST search against proteins deposited in the Protein Data Bank (PDB) with the amino acid sequence of Xcc17 ClpS revealed that E. coli ClpS (PDB ID 2W9R) has highest homology with the Xcc17 ClpS (56% Identities, 81% Positives). The predicted three-dimensional structure of Xcc17 ClpS using E. coli ClpS (PDB ID 2W9R) as template was shown in Fig. 3b. The amino acid residues contributed to the interaction of ClpS and the N-domain of ClpA in E. coli (Guo et al. 2002, Xia et al. 2004) are conserved in Xcc17 ClpS and are situated at P24, P25, Y28, E79, E82, and K84 (associated with contact A of ClpA), and D36 and Y37 (associated with contact C of ClpA) (Fig. 3b). These observations suggested that, similar to situation found in E. coli, the Xcc17 ClpS may complex with N-domain of ClpA and function as a modulator of ClpA.

The clpA gene is 2283 bp in length and is located at position 2227947–2230229 in the Xcc17 chromosome (GenBank accession number NZ_CP011946). It is predicted to encode an ATP-dependent Clp protease ATP-binding subunit ClpA, which consists of 760 amino acids with a calculated molecular mass of 83346 Da and a pI of 5.66. Domain analysis revealed that Xcc17 ClpA consists of three functional domains: one N-domain and two AAA+ ATPase domains (designated as AAA+ modules D1 and D2) which are each divided into a large sub-domain and a small sub-domain (Fig. 4a). A BLAST search against the PDB with the amino acid sequence of Xcc17 ClpA revealed that ClpA from E. coli (PDB ID 1KSF) has the highest identity with the Xcc17 ClpA (64% Identities, 79% Positives). The predicted Xcc17 ClpA three-dimension structure built by homology modeling using E. coli ClpA as temple structure is shown in Fig. 4b. In E. coli, the N-domain of ClpA has multiple ClpS binding sites (Xia et al. 2004) and is essential for docking of ClpS and required for the recognition, and degradation of some substrates (Dougan et al. 2002, Erbse et al. 2008, Guo et al. 2002, Zeth et al. 2002). Several amino acid residues contribute to the interactions of the N-domain of ClpA and the ClpS in E. coli (Xia et al. 2004) are highly conserved in Xcc17 ClpA. They include residues participating in ClpS binding via contact A (E23, F24, V27, E28, T81, R86, D116, and Y121) and residues involved in ClpS binding via contact C (S97, K99, N106, R130, and H139), respectively. In addition, three residues (H22, H29, and E63) in Zn²⁺ binding motif and two residues (Y14 and F84) in the N-domain hydrophobic path for peptide binding are also conserved in Xcc17 ClpA. These amino acid residues in the built three-dimension structure of ClpA are shown in Fig. 4c (left part). The ATPase domain of AAA+ protease consists of several classical key elements of ATPase function forming the nucleotide binding pocket, including Walker A, Walker B, sensor 1, arginine finger, and sensor 2 (Bittner et al. 2016, Miller and Enemark 2016, Wendler et al. 2012). The Walker A motif (GxxxxGKT/S, where x is any amino acid) is required for ATP binding and oligomerization whereas Walker B motif (hhhhDE, where h is any hydrophobic amino acid) is essential for ATP hydrolysis (Bittner et al. 2016, Miller and Enemark 2016). Both sensor 1 and arginine finger motifs are required for ATP hydrolysis whereas sensor 2 mediates conformational changes associated with a cycle of ATP binding and hydrolysis (Miller and Enemark 2016). These sequence motifs are highly conserved in both AAA+ modules D1 and D2 in Xcc17 ClpA. The Walker A motif in D1 and D2 is situated in 217–224 (GEAGVGKT) and 498–505 (GPTGVGKT), respectively. The Walker B motif in D1 and D2 is situated in 284–289 (VLFIDE) and 562–567 (VLLLDE). The sequences and positions of sensor 1, arginine finger, and sensor 2 motifs in D1 and D2 are as follows: (i) C³²¹IGSTT³²⁶ and L⁶⁰⁴VMTTN⁶⁰⁹ (sensor 1), (ii) R³⁴²R³⁴³ and R⁶⁴⁶ (arginine finger), and (iii) D³⁹⁴RLLPDKAID⁴⁰³ and M⁷⁰²GARPM⁷⁰⁷ (sensor 2). Extensive functional studies of different AAA+ proteins demonstrated that several amino acid residues are critical for AAA+ ATPase activity (Wendler et al. 2012). The Fig. 4c (right part) denotes the conserved nucleotide interacting residues in the AAA+ module of ClpA in Xcc17, in which the effects of mutations with regard to oligomerization and hydrolytic activity have been tested in other AAA+ proteins (Wendler et al. 2012). As denoted Fig. 4c (right part), the residues in AAA+ module D1 are: (i) K223 (Walker A), (ii) E289 (Walker B), (iii) T326 (sensor 1), (iv) R342 and R343 (arginine finger), and (v) R395 (sensor 2), and K504 (Walker A), D567 (Walker B), N609 (sensor 1), R646 (arginine finger), and R705 (sensor 2) in the AAA+ module D2. Given the conserved sequence motifs and catalytic residues of the X. campestris pv. campestris ClpA to homologs from a diverse set of bacteria, it is suggested that the Xcc17 ClpA is a functional AAA+ ATPase.

ClpA is required for survival under heat-shock condition

To investigate the function of ClpS and ClpA in X. campestris pv. campestris, we proceeded to generate the mutants (DclpS and DclpA) and their complementary strains DclpS(pRKclpS) and DclpA(pRKclpA) as described in the Materials and methods section. Due to the observations that the expression of both clpS and clpA is induced by heat shock, it is reasonable to suggest a role of these proteins in stress adaptation. We first characterized the impact of clpS and clpA deletions in sensitivity to heat stress. Cell suspensions from tenfold serial dilutions of the X. campestris pv. campestris wild-type strain, clpS and clpA mutant strains, and their complementary strains were spotted on the LB agar plates and incubated either at 28 ºC or 37 ºC. Under physiological temperature (28 ºC), all strains spotted at all densities grew similarly and displayed undistinguishable colony morphology (Fig. 5, left part). Under heat shock condition (37 ºC), the Xcc17(pRK415), DclpS(pRK415), DclpS(pRKclpS) and DclpA(pRKclpA) displayed similar growth behavior whereas the growth of clpA mutant DclpA(pRK415) was inhibited (Fig. 5, right part). It is suggested that deletion of clpS does not affect sensitivity to heat stress under the assay condition, whereas the clpA gene is required for stress tolerance in X. campestris pv. campestris. Similar situations are observed in the clpA mutants of B. susi and Salmonella Typhimurium (Ekaza et al. 2000, Sangpuii et al. 2018). In B. susi, the clpA mutant showed reduced growth rates at elevated temperature (Ekaza et al. 2000). In S. Typhimurium, the clpA mutant was hypersusceptible to 42 ºC exposure (Sangpuii et al. 2018). These observations contrast with results obtained for E. coli, in which the ClpA does not appear to be a heat-chock protein, and the clpA mutants grow well at temperatures between 25 and 42 ºC (Katayama et al. 1988). Despite the fact that E. coli ClpA is not a heat-shock protein, the clpA mutant is defective for growth at 46 ºC, and this protein appears to have a role in cellular recovery from transient incubation at 50 ºC (Thomas and Baneyx 1998).

The Clp family protease has a multitude of functions in bacteria, such as protein quality control, stress tolerance, and virulence factor expression (Malik and Brotz-Oesterhelt 2017). In S. Typhimurium, the clpA mutant strain showed susceptibility to HOCl (Sangpuii et al. 2018). Recently, it is reported that clpS and clpA are important in enabling E. coli to grow during H₂O₂ stress (Sen et al. 2020). In X. campestris pv. campestris, ClpX and ClpP, other members of Clp family protein, are documented to be important for the survival of this bacteria under various stresses, including temperature and puromycin (Li et al. 2020, Lo et al. 2020). The X. campestris pv. campestris ClpX also has a role in SDS tolerance (Lo et al. 2020). To examine whether clpS and clpA have a role in other stress tolerance, the sensitivities of DclpS and DclpA to H₂O₂, SDS, and puromycin were assessed. The DclpS and DclpA did not differ from the Xcc17 in susceptibility to these stresses (data not shown). It is similar to the observation in Acinetobacter baumannii, in which deletion of clpS and clpA genes does not alter oxidative stress sensitivity (Belisario et al. 2021). Taken together, these results suggested that clpX is more important than clpA in the survival of X. campestris pv. campestris under H₂O₂, SDS, and puromycin treatment. We cannot exclude that ClpS and ClpA contribute to other stresses, further insight regarding this aspect has to be gained.

In AAA+ proteins with two AAA+ ATPase modules, such as ClpA, the contributions of both regions to ATP biding/hydrolysis differ (Bittner et al. 2016). Mutational and functional analyses have demonstrated that the two domains of ClpA of E. coli possess different functional roles: first AAA+ domain in ClpA is crucial for oligomerization while the second AAA+ domain is primarily responsible for ATP hydrolysis (Pak et al. 1999, Seol et al. 1995, Singh and Maurizi 1994), and the two domains operate independently even in the presence of ClpP or ClpS (Kress et al. 2009b). In E. coli ClpA, it is demonstrated that: (i) the domain 1 mutant (ClpA-K220Q) was unable to form a hexamer, whereas the comparable domain 2 mutant (ClpA-K501Q) associated into a hexamer, and (ii) ClpA-K220Q was defective in ATPase activity and in the ability to activate protein and peptide degradation by ClpP, and (iii) ClpA-K501Q had very low ATPase activity and a sever defect in activation of protein degradation, but it was able to activate ClpP to degrade a peptide (Singh and Maurizi 1994). To investigate whether the conserved motif observed in ClpA had any impact in the heat stress tolerance in X. campestris pv. campestris, we performed site-directed mutagenesis to construct mutated versions of ClpA. The most common mutated residues (K223 in AAA+ module 1 and K504 in module 2) were selected and exchanged to glutamine. As depicted in Fig. 5 (left part), the DclpA complemented with mutated version pRKclpAK223Q or pRKclpAK504Q did not show significantly growth difference to other tested strains at 28 ºC. However, introduction of pRKclpAK223Q or pRKclpAK504Q to DclpA cannot restore the growth behavior as those observed in DclpA complemented with the wild-type version pRKclpA under heat shock condition Fig. 5 (right part). It is suggested that the putative residues K223 and K504 are associated with full function of ClpA in X. campestris pv. campestris stress tolerance. The growth of clpA mutant complemented with pRKclpAK223Q was similar to those mutant complemented with pRK415 (empty vector), whereas it is worth to noted that growth was prominent in the areas spotted with 5 × 10⁸ cells of DclpA(pRK415), DclpA(pRKclpA), and DclpA(pRKclpAK223Q), but not in areas spotted with the same concentration of DclpA(pRKclpAK504Q) at 37 ºC (Fig. 5, right part). It is not clear why introduction of ClpA with K504 mutation led to the clpA mutant a failure of forming colony under heat stress treatment. Introduction of pRKclpAK504Q seems to be detrimental to X. campestris pv. campestris and both predicted AAA+ modules in ClpA seems to possess different roles in this bacteria. The impacts of pRKclpAK504Q in the growth of clpA mutant and the function of each module of ClpA require further study.

The clpA mutant shows differential protein expression at high temperature

ClpA can function as a molecular chaperon preventing aggregation, and has a role in refolding and remodeling of proteins (Hoskins et al. 2001, Pak and Wickner 1997, Suzuki et al. 1997, Wawrzynow et al. 1996, Wickner et al. 1994). The impact of growth in the clpA mutant at 37 ºC suggested the participation of ClpA in preventing the accumulation of heat-inactivated and aggregate proteins in X. campestris pv. campestris. To test whether clpA deletion would alter the protein expression profile and affect the aggregation of proteins in heat-shocked cell, we grown the mutant and parental strains at 28 ºC or 37 ºC, then the protein samples were prepared and fractionated by SDS-PAGE as described in the Material and methods section. When cultures were grown at 28 ºC, the protein profiles obtained without Triton X-100 treatment were not significantly different between the parental strain and the clpA mutant (Fig. 6, lanes 1 and 5), similar patterns were observed when Triton X-100 treatment was included (Fig. 6, lanes 2 and 6). When the bacteria were cultured at 37 ºC, several protein bands differ between the parental strain and the clpA mutant, despite wholesale aggregation of proteins does not occur in the clpA mutant. In the absence of Triton X-100 treatment, the band I was observed in wild-type Xcc17 and was missing in the clpA mutant DclpA (Fig. 6, lanes 3 and 7). When Triton X-100 was included, the band II was observed in Xcc17 but not in DclpA, and bands III and IV were observed in DclpA, which were too faint to be visible in Xcc17 (Fig. 6, lanes 4 and 8). Further studies are required to identify these mainly altered protein bands to elucidate the significance of the change in their amounts. In E. coli, the amount of protein aggregation was not significantly increased in clpA mutant compared to the wild-type cells (Dougan et al. 2002), whereas in S. Typhimurium, the clpA mutant revealed greater amounts of protein aggregates than in the wild-type strain (Sangpuii et al. 2018).

ClpA is required for full virulence

As Clp family proteins are documented to be important for bacterial pathogenesis is several bacterial species (Brotz-Oesterhelt and Sass 2014), we wished to test whether this was also the case in X. campestris pv. campestris. To determine whether clpS and clpA is involved in the pathogenicity, the virulence of X. campestris pv. campestris was tested on the host plant cabbage by leaf-clipping inoculation. The inoculants for the pathogenicity test included Xcc17(pRK415), DclpS(pRK415), DclpS(pRKclpS), DclpA(pRK415), and DclpA(pRKclpA). The lesion length caused by the wild-type strain was approximately 2 cm at 14 days post inoculations, and the clpS mutant and its complementary strain caused virulence symptoms similar to those of the wild-type (Fig. 7). Although the clpA mutant could cause disease it was significantly attenuated compared with the wild type, and the complemented strain restored disease symptoms toward the wild-type phenotype (Fig. 7). These results indicate that clpS is not related to virulence and clpA is required for the full virulence of X. campestris pv. campestris. It is similar to the observations reported in Pseudomonas aeruginosa, in which clpA mutant but not clpS mutant exhibits virulence-attenuated phenotypes (Feinbaum et al. 2012). It has been suggested that disruption of ClpA function is responsible for the altered pathogenesis of Ralstonia solanacearum (Lin et al. 2008). A recent study in A. baumannii found that both clpS and clpA were important for virulence (Belisario et al. 2021).

In Xanthomonas species, it is reported that successful infection often depend on an arsenal of virulence factors, such as adhesins, degradative enzymes, and extracellular polysaccharides (Buttner and Bonas 2010, Chan and Goodwin 1999, Denance et al. 2016, Dow et al. 2003, Tang et al. 2021). To test whether the reduced virulence in the clpA mutant was correlated to a reduced production of these pathogenicity factors, we investigated the contribution of clpA to bacteria attachment and extracellular enzyme production. No considerable differences in both bacterial attachment and extracellular enzyme production were observed between the clpA mutant and the parental strain (data not shown). In A. baumannii, both clpS and clpA were essential for biofilm formation (Belisario et al. 2021). In X. campestris pv. campestris, clpX was also reported to be required for bacterial attachment (Lo et al. 2020). According to the X. campestris pv. campestris genome annotation, numerous virulence determinants are associated with bacterial pathogenesis, clpA might impact in other unidentified factor(s) which remains further evaluation.

Together with above results concerning ClpS, it is observed that mutation of clpS has no effect on stress tolerance and virulence, even its expression is heat inducible. Whether this gene has other physiological role(s) in X. campestris pv. campestris remains to be investigated. In the case of ClpA, even mutation of this gene resulted in growth defect at high temperature and attenuated pathogenicity, the virulence-related factors and a range of stresses did not alter significantly. Compared to the previous finding regarding ClpX, the other member of Clp ATPase, mutation of clpX caused pleiotropic effects, including attachment, virulence, and stress tolerance (Lo et al. 2020). It is reasonable to suggest that ClpX plays a more essential role in X. campestris pv. campestris. Owing the observations that (i) clpA expression is heat inducible, (ii) mutation of clpA is impact in growth at high temperature, and (iii) clpA mutant presents different protein pattern under heat treatment, it could be that ClpAP protease evolved to deal with the increased need for proteolysis caused by increased denaturation or aggregation, under heat shock conditions. It remains unknown whether ClpA can associated with ClpP, also the role of ClpS as an modulator affecting the function of ClpA is still unclear in X. campestris pv. campestris. Further clarification the nature of the ClpS, ClpA, and ClpP interaction can provide novel insights and gain a more comprehensive description of these proteins.

In this study, we investigated the transcription and function of clpS and clpA in X. campestris pv. campestris. First, upstream region analysis together with reporter assay indicated that (i) putative s³²-dependent sequences are present in the upstream region of clpS and clpA, (ii) both clpS and clpA contain their own promoter and are transcript independently, and (iii) the clpS and clpA promoters are induced approximately two-fold over control levels in response to heat stress. Second, through sequence comparison and homology modeling, the conserved sequence motifs were identified and the predicted three-dimensional structures of ClpS and ClpA were determined. Third, by combining insertional inactivation and phenotypic evaluation, it was revealed that mutation of clpA but not clpS causes defect in heat stress tolerance and pathogenicity, and the conserved residues of Walker A motif of the AAA+ ATPase module of ClpA are demonstrated to be required for ClpA function in heat tolerance. Finally, SDS-PAGE analysis revealed that the clpA mutant shows different protein profiles in response to heat shock treatment.

The Clp family proteins are found in most domains of life and the functional importance of these proteins varies from organism to organism. The genome of X. campestris pv. campestris is predicted to encode several common Clp family proteins, such as ClpS, ClpA, ClpX, ClpP, ClpQ, and ClpY (da Silva et al. 2002, Liu et al. 2015, Qian et al. 2005, Vorholter et al. 2008). While these proteins have been well-described in other organism, the function of these proteins are poorly understood in X. campestris pv. campestris. To date, only two (ClpX and ClpP) were found in the literature documenting their function in X. campestris pv. campestris. Both, ClpX and ClpP, are important in extracellular enzyme production, virulence, as well as heat and puromycin tolerance, suggesting the importance of ClpXP in pathogenesis and environmental adaptation (Li et al. 2020, Lo et al. 2020). The clpX mutant of X. campestris pv. campestris also exhibits reduced bacterial attachment and results in increased sensitivity to SDS (Lo et al. 2020). The expression of X. campestris pv. campestris clpP is shown to be heat inducible (Li et al. 2020). Combine with the finding we have presented here, there is a growing body of evidence that the Clp family proteins can be regarded as key virulence factors in X. campestris pv. campestris. They are plausible candidates for the development of new therapeutics to combat this phytopathogen.

Funding: 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)

Conflicts of interest/Competing interests: The authors declare no competing interests

Availability of data and material: Not applicable

Code availability: Not applicable

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Table 1. PCR primers used in this study

Primer	Sequence^a	Directionand purpose^b
35PstI	5¢-CTCGAGCAGGCTTTCGTCCGGCT-3¢	F, Promoter analysis
476XbaI	5¢-TCTAGATTATACGGTCCCGCGCCCTC-3¢	R, Promoter analysis
401HindIII	5¢-AAGCTTCCGGCCGGAGAACAGGCGTA-3¢	F, Complementation
946XbaI	5¢-TCTAGATGGTAACTCCGAACGGCTGT-3¢	R, Promoter analysis, mutant construction and complementation
621PstI	5¢-CTGCAGCAGTTCTTCAACCTGGA-3¢	F, Promoter analysis and mutant construction
899HindIII	5¢-AAGCTTCCCAACAAAAGCCGCATATTG-3¢	F, Mutant construction and complementation
3232XbaI	5¢-TCTAGACGATCAATCAACCGTAGCCG-3¢	R, Mutant construction and complementation
223F	5¢-ccggcgtgggccagaccgcgatc-3¢	F, site-directed mutagenesis
223R	5¢-gatcgcggtctggcccacgccgg-3¢	R, site-directed mutagenesis
504F	5¢-gaccggtgtgggccagaccgaggtcac-3¢	F, site-directed mutagenesis
504R	5¢-gtgacctcggtctggcccacaccggtc-3¢	R, site-directed mutagenesis

^a: Added restriction enzyme sites are underlined. The mutated bases are in boldface and underlined

^b: F, forward direction, R, reverse direction

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published 23 Mar, 2022

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Reviews received at journal
11 Aug, 2021
Reviewers invited by journal
08 Aug, 2021
Editor assigned by journal
02 Aug, 2021
First submitted to journal
01 Aug, 2021

You are reading this latest preprint version

Expression and Function of clpS and clpA in Xanthomonas Campestris Pv. Campestris

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results And Discussion

Conclusion

Declarations

References

Tables

Status:

Journal Publication

Version 1