5.2. Herramientas utilizadas
5.4.2. Variable Facilidad de Usabilidad
Endo-polygalacturonase (PG) activity is found in Ecc and Eca but not in Echr (George et al. 1991; Lei et al. 1985b; Plastow et al. 1986; Ried and Collm er 1986; Roberts et al. 1986a; Roberts et al. 1986b; Zink and Chatteijee 1985). Endo-PG hydrolyzes internal a-l,4-glycosidic bonds in pectate polymers (Nasuno and Starr 1966) generating oligomers o f galacturonic acid ranging from dimers to multimers (Roberts et al. 1986b; Willis et al. 1987). Echr, however, does possess an exo-PG (Brooks et al. 1990; He and Collmer 1990), which, using marker exchange mutagenesis, was shown to play a significant role in utilization o f polygalacturonate and induction o f PL (He and Collmer 1990). M ore recent work, using P L ' mutants of Echr EC 16, suggests that this enzyme (encoded by the pehX gene) does not contribute to the macerating activity o f Echr (Brooks et al. 1990). Unlike the PLs, PG does not require C a ^ + for activity. PG is constitutively expressed in Ecc (Chatteijee et al. 1981), its synthesis is not induced by pectate and is not affected by catabolite repression (Hinton et al. 1990). PG encoding genes (peh) have been
cloned from three different strains of Erwinia carotovora (Saarilhati et al. 1990a; Willis et al 1987; Zink and Chatterjee 1985), including SCRI193 (Plastow et al. 1986) and one strain o f Eca (Lei et al 1985b). In EC14 and £ c c l7 the peh gene has been shown to be linked to a pel gene (Roberts et al. 1986b; Willis et al. 1987). The two activities have been separated by insertion m utagenesis (Willis et al. 1987). Unlike the exo-PG o f Echr, the purified endo-PG o f Ecc and Eca are capable of causing potato tuber maceration (Lei et al. 1985b; W illis et al. 1987). The peh gene of Ecc SCRI193 shows 86% sequence homology to that o f other Ecc peh genes (Hinton et al. 1990). Recent work by George et al. (1991) has shown that Eca produces two PGs, one of pi 10.7 located in the extracellular fraction, and one o f pi 3.9 located mainly in the extracellular fraction with a small amount also occuring in the periplasm.
1 . 2 . 2 . 1 . 1 . C Pectin lyase
Most soft-rot erwinias produce endo-pectin lyase w hen induced by the DNA damaging agents mitomycin C, nalidixic acid and ultraviolet light (Itoh et al. 1980; Tsuyumu and Chatterjee 1984). Pectin lyase (PN L) cleaves pectin or methylesterified PGA, but is not active on PGA (Itoh et al. 1982; Kamimiya et al. 1974). PNLs from several strains of Ecc have been isolated which have optimum activity at a pH around 8.0., a pi o f 9 .6 and are immunologically identical (Itoh et al. 1982). PNLs from several strains o f Echr and Ecc w ere recognised by antibodies raised to an Echr PNL (Tsuyumu and Funakubo 1985). PNLs are immunologically distinct from PLs (Itoh et al. 1982) but pnlA has a 20% sequence homology with pelB o f Eca. a 25.1% homology with pelA o f Eca and a 28.5 % homology with pelE from Echr 3937 (Chattetjee et al. 1991).
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1 .2 .2 .1 .2 Regulation o f pectinase synthesis
Polygalacturonate (PGA) and its degradation products are inducers o f PL activity in the soft rot erwinias, as are plants (Garibaldi and Bateman 1971; Pupillo et al. 1976) and some plant extracts (Tomizawa et al. 1970; Tsuyum u 1977). Other factors also play a part in regulating pectolytic enzyme production, for example the production o f PLs by Eca SCRI1043 are reduced 3-6 fold at 3 0 .5 °C compared to 2 7 °C (Lanham et al. 1991; Salmond pers.comm .). This tem perature dependent production almost certainly has an affect on the pathogenicity and world wide distribution o f the species. The current knowledge o f regulation is given below.
1 .2 .2 .1 .2 .a Inducers
Induction levels o f the pel genes in Echr 3937 and B374 have been studied using pel-lacZ fusions in Lac" strains (Diolez and Coleno 1985; Hugouvieux-Cotte-Pattat et al. 1986; Hugouvieux-Cotte-Pattat and Robert-Baudouy 1985; Reverchon and Robert-Baudouy 1987). Expression of the 6-galactosidase gen e under the pel prom oter allows transcription from that promoter to be quantified. Levels o f transcription from the pel genes induced by PGA can vary. P L a /d /e synthesis in B374 varies between three and six fold (Reverchon and Robert-Baudouy 1987) w hile PLc in 3937 is increased by 37 fold on induction (Diolez an d Coleno 1985). On induction o f the pel genes all other genes in the pectinolysis pathw ay (Fig. A4) are also induced with intermediates in this pathway being the tru e inducers. These interm ediates have been identified as 2-keto-3-deoxygluconate (K D G ) (Hugouvieux- C otte-Pattat and Robert-Baudouy 1989), 2,5-diketo-3-deoxygIutarate (DKII) and possibly 5-keto-4-deoxyuronate (DKI) (Condemine et al. 1986).
POLYGALACTURONATE 2 -K E TO -3 -D E O X Y -G L U C O N A TE (K D G )
¿,0
2 -K ETO -3-D EO X Y -6 -P H O S P H O G L U C O N A TE (K PD G )I11
PYR U V A TE + TR IO S E-3-P H O S P H A TEFigureA4. Major pathways of pectate catabolism In bacteria. En zym e s for the catabolic steps are: 1) polygalacturonase, 2) pectate lyase, 3) oligogalacturonate hydrolase, 4) oligogalacturonate lyase, S) uronate isomerase, 6) altronate oxidoreductase, 7) altronate hydrolase, 8) 4-deoxy-L-threo-S-hexoseulose uronate isomerase, 9) 3-deoxy-D-glycero-2,5-hexodiulosonate dehydrogenase, 10) 2-keto-3-deoxygluconate kinase, 11) 2-keto-3-deoxy-6- phosphogluconate aldolase (from Chatterjee e ta l. 1985)
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1 .2 .2 .1 .2 . b Genetic control of regulation
1.2.2.1.
2. bi Catabolile repression
The synthesis of PL isozymes in Ecc and Echr is reduced in the presence o f a more easily catabolized carbon source than PGA, ie. synthesis is subject to catabolite repression (Fig A5) (Moran and S tarr 1969). The breakdown products o f PGA are also ab le to regulate PL production leading to a "self-catabolite" repression (C ollm er and Bateman 1981; Tsuyum u 1979). Such an effect is supported by the production o f mutants deficient in cyclic-AMP which have reduced PL synthesis (M ount et al. 1979), and the fact that the addition o f cAMP to Ecc suppresses the action o f repression (Hubbard et al. 1977). Further indirect support for the existence o f catabolite repression includes the presence o f a cya gene in Echr, encoding adenylate cyclase (Hedegaard and Danchin 1985). In the cri mutant o f Echr B374 (Hugouvieux-Cotte-Pattat et al. 1986) PL synthesis was less sensitive to the repression observed in the presence o f glucose in the wild type strain, as were cellulase and protease (Reverchon et al. 1990). The cri gene therefore seems to act at the level o f catabolite repression. Expression from pelB and pelC were more sensitive to the effects of this mutation than were pel A, pelD and pelE (Hugouvieux-Cotte-Pattat et al. 1986; Tsuyumu and Chatteijee 1984). Sequence hom ology to the catabolite activator protein (CAP) target site o f E.coli has been found in the promoter regions o f pelB and pelE o f Echr and pelC of Ecc SCRI193 (Hinton 1989b). In addition to catabolite repression, PL regulation is affected by a host o f regulatory genes. Below is a summary o f the regulatory genes o f Echr isolated to date.
Glucosa inhibits Adenylate cyclase Glucose stimulates
□
D A T P □ °FlgureAS Diagrammatic representation of the role of cyclic adenosine monophosphate (cAMP) and the catabollte activator protein (C A P ) In catabollte repression. In the case of catabollte-represslbte enzymes, binding of RNA polymerase only occurs If the CAP protein has first bound. This allosteric protein only binds If It has first bound cAM P which Is produced from ATP by the enzyme adenylate cyclase. T h e presence of glucose either Inhibits cAM P formation or stimulates Its breakdown. In the presence of glucose a deficiency of cAMP occurs preventing C A P binding and in turn preventing RNA polymerase binding.
kdgR and negative control of the pectinolvtic pathway
The kdgR gene o f Echr 3937 is a negative regulator o f the 2-keto-3-deoxygluconate transport system (Fig. A4) (Condem ine and Robert-Boudouy 1987; Reverchon et al. 1991). W hen inactivated by transposon mutagenesis (kdgR::Tn5) it leads to constitutive expression of all genes in the pectinolysis pathw ay, pelD and pelE being more strongly affected than pelA, pelB and pelC (Condom ine and Robert-Baudouy 1987; Reverchon and Robert-Baudouy 1987).
pecS and negative control of PL synthesis
Using M u/ac insertions in Echr 3937 Reverchon et al. (1990) isolated a pecS regulatory mutant with derepressed PL levels, which remained PGA inducible. No other genes involved in pectinolysis were effected. In contrast to kdgR, pecS mutations did not affect expression o f other genes in pectinolysis. Electrofocusing o f PL isozymes revealed that P L b and PLc strongly increased in pecS mutants whereas PLa, PLd and PLe w ere not o r were weakly affected by this mutation. pecS therefore appears to be a second negative regulatory gene whose product specifically represses pel genes.
gpiR and negative control of PL synthesis
Wild type Echr B374 synthesizes PLs at the end o f log phase but chemically- induced mutants affected in th e gpiR gene, synthesize PLs constitutively throughout the growth phase (Hugouvieux-Cotte-Pattat et al. 1986; Reverchon and Robert- Baudouy 1987). This probably results from the loss o f a control mechanism regulating the temporal activation o f PL and cellulase (Reverchon et al. 1990).
Unlike kdgR mutants, only pel genes in the pectinolysis pathway are affected, particularly pelD and pelE, while pelA expression is hardly affected (Reverchon and Robert-Baudouy 1987). Mutations in gpiR also enhance cellulase gene expression in Echr B374 (Hugouvieux-Cotte-Pattat et al. 1986). T h e gpiR mutation maps at a different location to the pel genes on the Echr B374 chromosome and the gene product may be a repressor (Reverchon and Robert-Baudouy 1987). Mutants constitutive fo r expression o f all pel genes have been isolated by Diolez et al. 1986 by marker exchange mutagenesis. These mutants, w hich may be gpiR mutants, produce the same amount o f PL in the presence or absence o f an inducer.
pecR and negative regulation o f PLa
Although the kdgR and gpiR genes appear to have little affect on the expression of pelA, a spontaneous mutant o f an Echr strain carrying a pelA-lacZ fusion has been isolated which leads to a constitutive B-galactosidase activity (Pupillo et al. 1976). In the absence o f an inducer pelA-lacZ is expressed 100-300 fold higher than the wild type (Reverchon and Robert-Baudouy 1987). This mutation has little effect on the other p el genes. As with the gpiR gene the pecR gene maps away from the affected gene and may therefore produce a repressor o f pelA.
Yankovsky et al. (1989) identified a mutation in a g e n e from Echr ENA49 they designated ptlR. Such a mutation led to constitutive expression of ptlA (equivalent to pelA) in the absence o f an inducer. This gene therefore appears to be a negative regulator o f ptlA and may be functionally equivalent to pecR.
pccYa
nd negative regulation of EME
Use o f Mu-lacZ inserts in Echr 3937 have led to the identification o f a gene ipecY) which appears to negatively regulate the expression o f pectin methylesterase
3 0
(Boccara and Chatain 1989). The relationship o f pecY to kdgR and peel is unclear.
peel affects induction o f pectinolvtic enzymes
In Echr 3937 regulation o f PLs and PME has been shown to be affected b y a further regulatory gene (peel) (Hugouvieux-Cotte-Pattat and Robert-Baudouy 1989). Mu- lac insertions in the gene peel lead to constitutive expression o f PLs and PM E in the absence o f an inducer, and a very high induction o f PL and PM E in th e presence of PG A.
1 .2 .2 .1 .2.biii Positive regulation
pecA and pecL in positive control o f PL synthesis
Spontaneous mutants o f Echr B374 with pelE-lacZ or pelD-lacZ fusions have been isolated with reduced B-galactosidase activity in the presence o f PGA or galacturonate. The relevant mutations (pecA") lead to reduction in the expression o f all pel genes in this strain and the corresponding gene may therefore encode a positive regulator. Regulatory mutants with decreased PL synthesis hav e also been obtained from strain 3937 by screening for Mu-/oc insertions in th e presence of PG A (Hugouvieux-Cotte-Pattat and Robert-Baudouy 1989) These m utants (pecU) showed a phenotype very similar to pecA mutants. Whether pecA a n d pecL are sim ilar or different genes is presently under investigation (Reverchon et al. 1990). Positive regulation o f pel genes by pecA and pecL may work in conjunction with the negative regulatory genes kdgR and pecS.
pecX and positive regulation o f PLa
gene (pecX) whose product seems to specifically activate pelA expression. Insertions in this gene result in a strong decrease in PLa synthesis, suggesting that pecX is involved in positive regulation o f pelA.
In conclusion the isolation o f regulatory mutants has shown the existence o f three types o f regulation affecting PL synthesis (catabolite repression, growth phase dependence and induction by PGA derivatives). In the regulation o f PL synthesis there appears to be a complex regulatory circuit with various regulatory genes, the expression o f which can vary, and which are probably involved in activating or repressing each o ther (Reverchon et al. 1990).