Vol. 174, No. 8

BACTERIOLOGY, Apr. 1992, p. 2679-2687

0021-9193/92/082679-09$02.00/0 Copyright C) 1992, American Society for Microbiology

Cloning and Characterization of a Gene Required for the Secretion of Extracellular Enzymes across the Outer Membrane by Xanthomonas campestris pv. Campestris NIEN-TAI


HUI-YUAN HUANG,1§ AND CHEN-YEU WU1"2 Agricultural Biotechnology Laboratories, 1 Graduate Institute of Botany,2 National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 40227, Republic of China Received


November 1991/Accepted 6 February 1992

Nonpathogenic mutants of Xanthomonas campestris pv. campestris, generated from transposon mutagenesis, accumulated extracellular polygalacturonate lyase, at-amylase, and endoglucanase in the periplasm. The transposon Tn5 was introduced by a mobilizable, suicidal plasmid, pSUP2021 or pEYDG1. Genomic banks of wild-type X. campestris pv. campestris, constructed on the broad-host-range, mobilizable cosmid pLAFRi or pLAFR3, were conjugated with one of the mutants, designated XC1708. Recombinant plasmids isolated by their ability to complement XC1708 can be classified into two categories. One, represented by pLASC3, can complement some mutants, whereas the other, represented by a single plasmid, pLAHH2, can complement all of the other mutants. Restriction mapping showed that the two recombinant plasmids shared an EcoRI fragment of 8.9 kb. Results from subcloning, deletion mapping, and mini-Mu insertional mutation of the 8.9-kb EcoRI fragment suggested that a 4.2-kb fragment was sufficient to complement the mutant XC1708. Sequence analysis of this 4.2-kb fragment revealed three consecutive open reading frames (ORFs), ORF1, ORF2, and ORF3. Hybridization experiments showed that Tn5 in the genome of XC1708 and other mutants complemented by pLASC3 was located in ORF3, which could code for a protein of 83.5 kDa. A signal peptidase II processing site was identified at the N terminus of the predicted amino acid sequence. Sequence homology of 51% was observed between the amino acid sequences predicted from ORF3 and the pulD gene of Klebsiella species. In gram-negative bacteria, the extracellular proteins have

the periplasm was transported across the outer membrane as soon as the repressedpul genes were induced. Thus, thepul genes are probably required for the secretion of extracellular pullulanase across the outer membrane. X. campestris pv. campestris, the causal agent of black rot in the crucifers, is another of the relatively few gramnegative bacteria that are known to secrete enzymes extracellularly. The enzymes exported include ot-amylase (46), polygalacturonate lyase (7), protease (7, 44), and endoglucanase (18). One class of the nonpathogenic mutants isolated by Daniels et al. (7) were defective in the secretion of extracellular enzymes (11). Complementation of this mutant was achieved with a gene cluster (47). Similar observations were made in this study. One group of the TnS mutants produced from this study was complemented with a 4.2-kb DNA fragment, the sequence of which revealed three successive open reading frames (ORFs) encoding for proteins with molecular weights of 26,000, 28,000, and 83,500, in that order. The results from hybridization experiments using different parts of this 4.2-kb DNA fragment as probes indicated that TnS in these mutants was inserted in the gene encoding the 83.5-kDa protein. The predicted amino acid sequence of this protein appeared similar to that of the pulD gene of Klebsiella species (9).

two lipid bilayers to cross, in contrast to the proteins targeted to the periplasm or the outer membrane. Depending upon the secretion route and the genes involved, two major

secretion pathways can be identified. Without traversing the periplasm, oc-hemolysin of Escherichia coli is secreted through a hemolysin translocator composed of HlyB, HlyD (24), and ToiC (48). Two genes, prtD and prtE, required for the secretion of an extracellular protease by Erwinia chrysanthemi were found to be homologous to hlyB and hlyD, respectively (27). In the second category, extracellular proteins, for example, the pullulanase of Klebsiella oxytoca, are secreted via the periplasm (34). A closely linked operon includes at least 12 pul genes encoding proteins required for the secretion of pullulanase in addition to the sec genes involved in the translocation of proteins across the inner membrane (34, 35). Homologs of the pul genes are also required for the secretion of extracellular proteins by E. chrysanthemi (22), Pseudomonas aeruginosa (15), Xanthomonas campestns pv. campestris (12), and Yersinia enterocolitica (29). Mutations in these genes can result in the accumulation of extracellular proteins in the periplasm (1, 7, 11, 16, 29, 34, 45, 49). Recently, Pugsley et al. (36) have elegantly demonstrated that the pullulanase accumulated in * Corresponding author. t Present address: Union Chemical Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan, Republic of China.

Present address:

MATERUILS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. E. coli SM10 and plasmid pSUP2021 were given to us by R. Simon. The E. coli strain containing plasmid pEYDG1 was obtained from E. A. Yakobson. The plasmids pLAFR1 and pRK2013 were

of Microbiology and

Department Immunology, Chang Gung Medical College, Taoyuan, Taiwan, Republic of China. § Present address: Molecular Biology Division, Development Center for Biotechnology, Taipei, Taiwan, Republic of China. t





TABLE 1. Bacterial strains and plasmids

Strains X. campestris pv. campestris XC1701 XC1708 XC1704 XC1707 XC1715 XC17206 XC17433 XC17585 XC17601 XC17719 XC17721

Spontaneous mutant of XC17a This study This study This study This study This study This study This study This study This study This study


Rif' Kanr Xps Rifr Kanr XpsRif Kanr XpsRifT Kanr XpsRift Kanr XpsRifT Kanr XpsRifT Kanr XpsRifT Kanr XpsRifT Kanr XpsRifT Kanr Xps-

E. coli

SM10 BNN45 DH1 JM101 SY634 P0111734D M8820

Plasmids pSUP2021 pEYDG1 pLAFR1 pLAFR3 pRK2013 pLASC3 pLAHH2 pSC1810 a

Source or reference

Relevant characteristics

Strain or plasmid

recA, integrated RP4-2-Tet::Mu hsdR supE44 supF thi met recAl endAl gyrA96 thi-1 hsdR17 (rK- MK+) supE44 supE thi A(lac-proAB) F'[traD36 proAB+ lacIq lacZAM15] ara A(gpt-lac) argE(Am) gyrA rpoB thi polA34(ts) F- araD ara::(Mu cts) A(lacIPOZYA) galKE rpsL A(srl-recA)::Tn1O with Mu dII1734(Kmr lacZYA) F- araD A(ara-leu) A(proAB-argF-1acIPOZYA) rpsL

40 C.-H. Lee 20 J. Messing 43 4

Ampr Camr Tet::Tn5 (Kanr) Kanr ( Tn5 in oriT of RK2) Tra- Mob' Tetr IncP replicon Tra- Mob' Tetr IncP replicon Tra+ Mob' Kanr ColEl replicon X. campestris pv. campestris DNA cloned in pLAFR1 X. campestris pv. campestris DNA cloned in pLAFR3 8.9 kb from pLASC3, cloned in pUC18

40 51 17 41 14 This study This study This study


Bacterial strain obtained from S.-T. Hsu of the Plant Pathology Department of National Chung Hsing University.

kindly provided by S. R. Long. E. coli POII1734D and M8820 were kind gifts from M. J. Casadaban. Media. Luria broth (LB) medium (30) was used to grow E. coli and X. campestris pv. campestris. For preparing extracts for enzyme assays, the following media were used. The basal culture medium (4 g of yeast extract, 5 g of (NH4)2SO4, 1 g of KH2PO4, 0.5 g of MgSO4. H20, 0.1 g of CaCl2- 2H20, 2 ,ug of MnSO4. H20, 10 ,ug of ZnCl2, 1 ,ug of H3BO3, and 20 ,ug of FeCl3- 6H20 per liter [pH 6.8]) (32) supplemented with 0.5% sodium polypectate was used for the examination of polygalacturonate lyase, and XOL medium [0.7 g of K2HPO4, 0.2 g of KH2PO4, 1.0



(NH4)2SO4, 0.01 g of FeSO4 7H20, 0.001 g of MnCl2, and 0.1 g of MgCl2 per liter (pH 7.15)] (6) supplemented with 0.125% tryptone, 0.125% yeast extract, and 0.2% starch was used for the examination of a-amylase. The following antibiotics used in the selection of transconjugants were added at the following concentrations (in micrograms per milliliter): rifampicin, 100; kanamycin, 50; and tetracycline, 15. Pathogenicity test. In testing for pathogenicity, cabbage of the K-Y cross hybrid (by Takii & Co., Ltd., Kyoto, Japan) was inoculated at the tip of the leaves. A V-shaped cut was made by using flame-sterilized scissors dipped in cell suspensions of fresh colonies. Transconjugants from Tn5 mutagenesis were inoculated along with the parental strain as the positive control and saline solution as the negative control. It took 3 to 9 days for the typical black rot symptoms to

appear, depending on the time of inoculation and the amount of inoculum. Plants that repeatedly did not show symptoms within 5 to 7 days after symptoms appeared on plants inoculated with the parental strain were scored as nonpath-


Conjugation. Donor, helper, and recipient cells were mixed at a 1:1:10 ratio in 50 ,ul of LB. Matings were conducted on sterile nitrocellulose paper on solid medium incubated at 28°C overnight. At the end of incubation, cells from the paper were resuspended in 1 ml of LB and then plated at the appropriate dilutions on selective medium containing the appropriate antibiotics. Cellular fractionation. The supernatant obtained following centrifugation at 17,000 x g for 10 min was taken as the extracellular fraction. After being washed two times with an equal volume of H20, the cell pellet was treated on ice for 2 h with lysozyme (200 ,g/ml) in a solution made up of 20% sucrose-30 mM Tris-HCl (pH 8.0)-i mM EDTA. Then the lysozyme-treated cells were pelleted by centrifugation at 25,000 x g for 10 min. The supernatant from this centrifugation was collected as the periplasmic fraction. The cell pellet was resuspended in 10 mM Tris-HCl (pH 8.0) and was passed through a syringe with a 23-gauge needle and centrifuged again at 25,000 x g for 15 min. The supernatant was collected as the cytoplasmic fraction. Enzyme assays. Polygalacturonate lyase was assayed by measuring the increase in A232 as a result of the appearance

VOL. 174, 1992

of unsaturated galacturonide in 0.15 M glycine-NaOH buffer at pH 9.5 containing 1 mM CaCl2 (32). One unit of activity represents the release of 1 ,umol of unsaturated uronide per min per A550 unit at 30°C. a-Amylase was assayed by determining the concentration of reducing sugar (38) after the assayed fraction was incubated with 1% starch in the presence of 27.5 mM phosphate buffer (pH 7.15) at 28°C for 10 min. One unit is equivalent to 1 ,umol of maltose produced per minute per unit of cell density. Endoglucanase activity was detected on LB agar plates containing 0.5% carboxymethyl cellulose. After incubation, plates were stained with 0.1% Congo red for 30 min, rinsed with water, and washed twice with 1 M NaCl (18). 13-Lactamase was assayed by monitoring the decrease in the A230 of penicillin G (42). Malate dehydrogenase was assayed in 0.1 M phosphate-42 mM aspartate buffer, pH 7.4. Oxalacetate-dependent NADH oxidation was monitored as the decrease in A339 (2). Construction of genomic bank. Cosmid pLAFR1 or pLAFR3, prepared from CsCl-ethidium bromide centrifugation, was digested with EcoRI or BamHI, respectively, and then dephosphorylated with calf intestinal alkaline phosphatase according to the method of Maniatis et al. (28) for construction of a genomic bank. XC1701 total DNA partially digested with EcoRI was ligated to pLAFR1 and DNA partially digested with Sau3A was ligated to pLAFR3 at 4°C for 16 h by T4 DNA ligase. The ligated mixtures were then packaged in vitro and used to infect E. coli BNN45 and were then plated on LB agar containing tetracycline. Approximately 1,460 colonies appeared after incubation. They were pooled and saved in 50% glycerol at -20°C. Construction of deletions. The 8.9-kb EcoRI fragment was subcloned in pUC18. Deletions of various lengths from the left-end EcoRI site were created by following the procedure developed by Henikoff (23). Following digestion with HindIII, the ends were filled with a-phosphorothioate deoxyribonucleoside triphosphates (40 ,uM of each) (37) by using the Klenow fragment of E. coli DNA polymerase I at 37°C for 10 min to generate a protected end on pUC18. The plasmid was then digested with XbaI to create a 5' protruding end on the inserted DNA for further exonuclease III digestion. The exonuclease III digestion was performed in 15 ,ul of a solution containing 66 mM Tris-HCl (pH 8.0)-0.66 mM MgCl2. Forty units of exonuclease III (Promega) was included for 1.5 ,ug of DNA. Incubation at 37°C for 1 min followed by Si nuclease (12 U; Boehringer Mannheim Biochemicals) digestion at room temperature for 30 min generated an array of DNA fragments with deletions of up to 7 kb. S1 nuclease was then inactivated by heating at 70°C for 10 min. The ends were filled with deoxyribonucleotide triphosphates by using the Klenow fragment before T4 DNA ligase was included to circularize the deleted plasmids. Following transformation, ampicillin-resistant transformants were screened for plasmids with deletions of various lengths. Construction of mini-Mu insertions. The procedures of Castilho et al. (4) were followed for the construction of mini-Mu insertions. Phage lysates were prepared from E.

coli POII1734TR containing pSC1810 by raising a mid-logphase culture to 43°C for 30 min and then centrifuging it at 25,000 x g for 10 min. The supernatant, after being filtered through a 0.45-,um-pore-size filter, was mixed with E. coli M8820 Mu cts and incubated at 28°C for 1 h. Plasmids isolated from the transductants, which were selected on medium containing ampicillin and kanamycin, were examined with restriction enzymes to identify the mini-Mu insertions on the 8.9-kb fragment and to locate their insertional sites.



Complementation. All of the subcloning, deletion constructions, and mini-Mu insertions were conducted with pUC18-based clones. Following the constructions, plasmids prepared from the subclones and the deletion mutants were ligated with one of the two mobilizable, broad-host-range plasmids, pLAFR1 and pLAFR3, at the EcoRI site. The mini-Mu insertions in the 8.9-kb fragment were allowed to exchange with pLAHH2, which was pLAFR3 based, via homologous recombination in RecA+ E. coli JM101. Plasmids isolated from JM101 were then transformed into E. coli SY634, whose DNA polymerase I was temperature sensitive. The transformed SY634 plasmids, plated on medium containing kanamycin and tetracycline and incubated at 42°C, were all ampicillin sensitive. Examination of the plasmids with restriction enzymes indicated that they were indeed pLAHH2::mini-Mu. After the above constructions were made into pLAFR1 or pLAFR3, they were introduced into the mutant XC1708 via conjugation aided by the E. coli strain containing the helper plasmid, pRK2013. Transconjugants were then toothpicked onto plates of M9 supplemented with sodium polypectate. After they were incubated overnight, saturated copper acetate was overlaid for the detection of degradation of sodium polypectate (1). XC1701 and each secretion mutant were included as positive and negative controls, respectively. A clear zone surrounded by a precipitation zone formed only around the positive control and the complemented mutants. Southern blot hybridization. The procedures of Maniatis et al. (28) were followed for Southern blot hybridization. Radiolabelled probes were prepared from in vitro DNA synthesis with Klenow enzyme on multiprimer (13). DNA sequencing. The DNA sequence of the 4.8-kb fragment was obtained from three BamHI subclones and one EcoRI-BamHI subclone. Nested deletion clones of each subclone were generated from each end as described previously (23). Sequences were determined by using the dideoxy method of Sanger et al. (39). Sequences at the junctions between the subclones were confirmed by using synthetic oligomers based on sequences adjacent to the junctions. Mini-Mu insertional sites were determined by using oligomers according to the left-end sequence of Mu (19). Nucleotide sequence accession number. The nucleotide sequence reported in this article has been submitted to the GenBank Data Library and assigned accession number M81111. RESULTS Isolation of mutants. Two collections of mutants of X. campestris pv. campestris were obtained via the introduction of transposon TnS by using two mobilizable, suicidal plasmids, pSUP2021 and pEYDG1. Kanamycin-resistant transconiugants were generated at a frequency of 1 x 10' to 4 x 10 per recipient from pSUP2021 and a frequency of 2 x 10-8 to 5 x 10-7 per recipient from pEYDG1, the lowest frequency of which was at least 10 times higher than the spontaneous mutation rate. Mutants of various kinds, six auxotrophs with six different growth requirements (25), and three pigmentation mutants were obtained from 2,150 kanamycin-resistant transconjugants. In addition, when, except for the auxotrophs, the same group of transconjugants were tested on cabbage leaves, 10 were nonpathogenic. No symptoms were observed at the cut site on the leaves inoculated with the mutants, whereas the site inoculated with the parental strain clearly showed symptoms. When examined on plates, all of the nonpathogenic mutants exhibited simul-






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Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris.

Nonpathogenic mutants of Xanthomonas campestris pv. campestris, generated from transposon mutagenesis, accumulated extracellular polygalacturonate lya...
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