Letters in Applied Microbiology 1992, 14, 233-237

Effect ofpavB on plasmid stability and gene expression in

Xanthomonas campestris A N D R E AD E L I M AP I M E K T AY.. B . R O S A T O * & s. A S T O L t i - F I L H O ? Deptzrtment of Genetics and Ecolution, Institute of’Biology and C B M E G , U N I C A M P . 13081 Campinus, S P , Brazil, and +Department of Cellular Biology, Institute o f Biological Science. Unicersity of Brasilia, 70.760 Brasilia, D F , Bruzil FS/133: received 17 September 1991 and accepted 30 January 1993 PIMENTA A,. L . . R O S A T O , Y . B . & A S T O L F I - F I L H O S ., 1992. Effect o f p a r B on plasmid stability and gene expression in Xanthomonas campestris. Letters in Applied Microbiology 14. 233-237. The stabilization locus parB was subcloned into the broad host range plasmid pAP2, which contains the alpha-amylase gene from Bacillus subtilis, and introduced into Xanthomonas campestris pv campesrris and X.r.pv manihoris. Analysis of the stability of plasmid pAP2 (parB-) and pAP23 (parB*) showed that the parB locus decreased significantl) the plasmid loss rate mainly by X.c.pv campesrris. The lower efficiency of stabilization in X.c.p\ mariihotis was probably due to the incompatibility system between the native plasmids and the newlj introduced pA1’23. Although parB had conferred higher stability, it determined a lower rate of alphaamylase activity even by the strain Cm where its stabilization rate was higher.

Xunthomonas cumpesrris is a Gram-negative, phytopathogenic bacterium responsible for a large number of economically important plant diseases. It is also the producer of an extracelluIar polysaccharide known as xanthan gum. which has been largely used in petrochemical food and textile industries (Baird et a / . 1983). Due to the great interest in studying the mechanisms involved in both phytopathogenicity and xanthan gum production. vectors are being developed to enable genetic manipulations in X . campestris. The broad host range plasmid pMFY40 (Fukuda & Yano 1985) was used to subclone the alpha-amylase gene from Bacillus subtilis in X . campesfris (Stripecke er al. 1989). This plasmid (pAPl), when introduced in strains of X . campestris, conferred the ability to hydrolyse starch but showed genetic instability when grown without selective pressure. In this report we describe the introduction of the stabilizing locus parB (Gerdes 1988) in pAP2 (Pimenta e l ul. 1991) which differs from PAP1 only in the length and restriction sites of

* Corresponding author

any insertion. The genetic stability of this plasmid before and after the insertion of purB as well as its effects on the alpha-amylase gene expression in X . curnpesrris were analysed.

Materials and Methods STRAINS

Bacterial strains and plasmids used are listed in Table I . Strain C a l l 0 0 is a wild type. amylolytic and plasmidless, while Ma3280 is nonamylolytic and harbours three native cryptic plasmids (Stripecke & Rosato 1988). CULTI RE MEDIA A h D ( O \ D I T I O \ S

Composition of L and YM medium used for growth of Escherrchia ioli and X rampestric, respectively. and other procedures here carried out as reported by Stripecke et a / (1989) DNA MA\IPCLATIO\

Large scale preparations of plasmids and general procedures were carried out according

Andrea de Lima Pimenta et al.


Table 1. Bacterial strains and plasmids Strain or plasmid


Source or reference

X.c.pv campestris Ca 1100 Cm Cm2 Cm23

Wild type Ca 110 CmR CmJpAP2 Cm2JparB+

NRRL B-1459 Lanza & Rosato (1986) Pimenta et al. (1991) This study

X.c.pv manihotis Ma 3280 Nal Na12 Na123

Wild type Ma 3280 NalR Nal/pAPZ Nal2/parB+

Biological Institute, Brazil Stripecke et al. (1989) Pimenta et al. (1991) This study

F - , leuB6, proA2, recA13 thil, aral4, lacy 1, gnlK2 xy15, mtll, rpsL20, hsdS20

Boyer & RoullandDussoix (1969)

ColEl, Mob+, Tra', KmR pM [email protected]/am y pAP2/parB

Figursky & Helinsky (1979) Pimenta et al. (1991) This study

Escherichia coli HBlOl

Plasmids pRK2013 pAP2 pAP23



Cm, chloramphenicol; Nal, nalidixic acid; Ap, ampicillin; Km, kanamycin; amy, alpha-amylase gene; Tc, tetracycline; Mob, mobilizable for transfer; Tra, selftransmissible.

to Maniatis et al. (1982). For small scale preparations the alkaline lysis method was used (Birnboin & Doly 1979). Isolation of DNA fragments was performed with DEAE filters or from low melting point agarose gels. Restriction endonucleases, T, DNA ligase and bacterial alkaline phosphatase were used as recommended by the manufacturer.

selected for chromosome-encoded resistance (chloramphenicol or nalidixic acid). Samples were taken at 24 h intervals (three repetitions), and plated on medium containing the same antibiotic with and without addition of tetracycline (plasmid-encoded resistance).


Escherichia coli cells were transformed with plasmid DNA according to Mandel & Higa (1970). CONJUGATION

Triparental conjugation was carried out by mixing 3 ml of overnight cultures of donor, receptor and helper (pRK2013) strains. The mixture was centrifuged and the pellet was incubated overnight on a Millipore membrane placed on YM solid medium. PLASMID STABILITY

Plasmid stability was measured by growing cells in L medium containing the antibiotic which

Alpha-amylase present in supernatants of cultures was determined as described by Smith & Roe (1949). Total protein was measured according to Bradford (1976) and the specific activity of alpha-amylase according to Prudence et al. (1989). Three repetitions were used for each measurement and statistical analysis was performed by the Tukey test.

Results S U B C L O N I N GO F




pKG1022 was digested with PstI and the 1.63 kb fragment containing the parB (0.58 kb) and the ophA (1.05 kb) genes isolated. Plasmid pAP2 was linearized with PstI, dephosphorilated and ligated to the 1.63 kb parB/ophA frag-

Eflect of parl? in X.campestris P SI A1





x s


A1 Sl




0 0.58 1.63 2.33



3.93 4.28 5.51



Fig. 1. Restriction map of pAP23. (I) parB locus; (11) ophA gene; (111) amy gene; (IV) TcR.Restriction sites: A, Aual; AI, A c c I : B, BamH1; E, EcoRI; EV, EcoRV; H, HindlII: HC. Hincll: K. K p n l : P. PsrI; PV. P c l l ; S, S a d ; SI, SalI: X. XhaI.

ment. The ligation mixture was used to transform E. coli HBlOl and transformants were selected for kanamycin and tetracycline resistance (KmRand TcR), sensitivity to ampicillin and alpha-amylase production. Twelve colonies showing large halos of alpha-amylase activity and showing the required resistance genes were selected. The plasmids were extracted and characterized by restriction digests. One transformant, containing the 14.8 kb plasmid, showed the predicted pattern of digestion and was chosen for further experiments. This plasmid was named pAP23 (Fig. 1). Determination of the orientation of the parB.! ophA fragment was determined after digestion of pAP23 with EcoRI. According to Gerdes (1988) there is one EcoRI site between the parB and ophA genes and digestion of pAP23 with EcoRI showed two fragments of approximately 1.8 and 13.0 kb. It was thus concluded that the ophA gene was inserted in the orientation shown in Fig. 1. The new plasmid pAP23, as well as the original pAP2, were introduced into strain Cm of X.c.pv campestris and strain Nal of X.c.pv manihotis by triparental matings. S T A B I L I T Y OF





-me d Fig. 2. Plasrnid loss rate of pAP2 (parB ) and pAP23 @arB-) in strain Nal of X c pv manihotis (a) and strain Crn of X c pv campestris (b) (a) pAP23/Nal, H, pAP2/Nal (b) pAP23/Cm, 0, pAP2/Cm



first 24 h of incubation in non-selective medium the plasmid loss rate decreased but still pAP23 was significantly more stable than pAP2 in both strains. A L I'H A - A M Y L A S E E X P R E S S 10 h

The results obtained (Fig. 3) show that the highest production of alpha-amylase was detected in the Cm strains and peak production occurred around 27-30 h for all strains. After



The results obtained (Figs 2a,b) showed that pAP23 (parB+) is more stable than pAP2 (parB-) in X . campestris. This stabilization was clearly detected during the first 24 h of growth. During this period, the plasmid loss rate of pAP23 ranged from 4% (for Nal strain) to 2.7% (for Cm strain), while pAP2 was lost at a rate of 66% and 74% (Nal and Cm strains, respectively) during the same period. After the

9 Y





Fig. 3.

., 1


30 -ime




Cm2: t ,Cm 23: +. Nal 2: [3,Nal 23.


Andrea de Lima Pimenta et al.

this time there was a decreased value of specific activity. The differences in the specific activity was considered significative (Tukey test 5%, not shown) when Cm2 was compared with Cm23 and Na12 with Na123. The introduction of parB into plasmid pAP2 determined a significant reduction in the alpha-amylase activity by the two host strains, C m and Nal.

membrane. In this case other extracellular enzymes should be affected by the parB system. This hypothesis could be tested in relation to other enzymes.



The stability of plasmids is one of the required characteristics for ideal cloning vectors. Cornmonly the stabilization is controlled by adding antibiotics to the culture medium leading to selection of cells harbouring the plasmid. More recently the oarB locus has been used as a stabilization system in bacteria other than E. coli (Boe et a/. 1987; Gerdes, 1988). In X . carnpestris and other phytopathogenic bacteria a few cloning vectors are available and most of the genomic libraries are constructed in special cosmids like pLAFR' and pLAFR3 (Friedman et a / . 1982; Staskawicz et al. 1987). We have used pMFY40 as a cloning vehicle for amylase production and stabilization was achieved only using antibiotics (Stripecke et a / . 1989; Pimenta et al. 1991). The insertion of p a r B locus into this plasmid resulted in higher levels of maintenance in the Nal and Cm host strains (16 and 27 times higher, respectively). The monitoring of plasmid was done for long periods because of the time required in fermentation for xanthan gum production. Lower stabilization in Nal strain could be due to the existence of native cryptic plasmids (Stripecke & Rosato 1988) which might interfere with the replication system. However, an unexpected consequence of the insertion of parB was the observed decrease in the alphaamylase activity. In the Cm strain, the level of this inhibition was lower, probably due to the double alpha-amylase coding system, the chromosomal and plasmid genes. We do not know if both systems are being altered; however, in Nal strain, which owns only the plasmid system operating for amylase production the decrease is higher. There is no evidence to suggest how the parB exerts its influence in this inhibition, neither if this effect is restricted to the plasmid-borne genes. It is also possible that instead of inhibiting the alphaamylase production, the parB locus might be acting in the enzyme transport through the

BAIRD,J.K., SANDFORD, P.A. & CASTEL,I.W. 1983 Industrial applications of some new microbial polysaccharides. Biotechnology 778-783. BOE, G., GERDES,K. & MOLIN,R. 1987 Effects of genes exerting growth inhibition and plasmid stability on plasmid maintenance. Journal of Bacteriology 169.4646-4650. BIRNBOIN, H.C. & DOLY,J. 1979 A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleics Acid Research 7, 15131523. BOYER, H.W. & ROULLAND-DUSSOIX, D. 1969 A complementation analysis of the restriction and modification in Escherichia coli. Journal of Molecular Biology 41,459-472. BRADFORD, M.M. 1976 A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry 72,248-254. FRIEDMAN, A,, LONG,S., BROWN,S., BUIKEMA, N. & AUSUBEL, F. 1982 Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18, 289-296. FUKUDA, H. & YANO,K. 1985 Construction of broad host range cloning vectors for gram negative bacteria. Agricultural Biological Chemistry 49, 27192724. GERDES,K. 1988 The parB (hoklsok) locus of plasmid R1: a general purpose plasmid stabilization system. Biotechnology 6, 1402-1405. LANZA, S.A. & ROSATO, Y.B. 1986 Producao de goma xantana por mutantes e recombinantes de Xanthomonas campestris. Resumos da XI11 Reuniao Anual de Genetica de Microrganismos: 84. Ribeirao Preto, SP, B r a d MANUEL,M. & HIGA,A. 1970 Calcium-dependent bacteriophage DNA infection. Journal of Molecular Biology 53, 159-162. MANIATIS, T., FRITSCH,E.F. & SAMBROK, J. 1982 Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratories. PIMENTA,A.L., ROSATO,Y.B. & ASTOLFI-FILHO, S. 1991. Expression of a subcloned alpha-amylase gene under the control of a Xanthomonas campestris promoter. F E M S Microbiology Letters (in press). PRUDENCE, K.W., KATZ,A,, SUGRUE, J.A. & THOMPSON, J.A. 1989 The effect of glucose on the expression of a cloned Bacillus amyloliquefaciens alpha-amylase gene - on strain of Bacillus subtilis. Current Microbiology 18,27-31.

We thank CAPES for the fellowship to A.L. Pimenta, and FAPjUNlCAMP and FINEP/ ProBio for the financial support.

Effect of parB in X. campestris


STRIPECKE,R. & ROSATO. Y.B. 1988 Variability in the SMITH,B. & R o t . J 1949 A photometric method for electrophoretic pattern of alpha-esterases and plasthe determination of alpha-amylase in blood and mids in Xanthomonas campestris. Fitopatoloyia Braurine with use of the starch-iodine colour. Journal sileira 12, 358-36 I. of Biological Chemistry 179, 53-59. S. 1989 STASKAWICZ, B.. DAHLBECK,D.. KEEN.N. & N ~ P O L I , STRIPECKE,R., ROSATO,Y.B. & ASTOLFI-FILHO, Subcloning and expression of the alpha-amylase C. 1987 Molecular characterization of cloned avirugene from Bacillus subtilis in Xanthomonas camlence genes from race 0 and race 1 of Pseudomonas pestrrs. Applied Microhioloyj, and Biotechnology 31, syrinyae pv qlrcrnea. Journal of Bucferiologj, 169, 512-517. 5789--5794.

Effect of parB on plasmid stability and gene expression in Xanthomonas campestris.

The stabilization locus parB was subcloned into the broad host range plasmid pAP2, which contains the alpha-amylase gene from Bacillus subtilis, and i...
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