Control by cAMP-CRP complex of the expression of the PTS-dependent Erwinia chrysanthemi clb genes in Escherichia coli

Abstract Influence of the crp, cya and ptsIH gene products on the functioning and/or regulation of the Erwinia chrysanthemi clb genes was studied in Escherichia coli . Transport of cellobiose was found to be mediated by the phosphotransferase system and the expression of the clb genes to be positively controlled by the cAMP-CRP complex. We postulate that the same situation may occur in E. chrysanthemi .     

Analysis of the Erwinia chrysanthemi arb genes, which mediate metabolism of aromatic beta-glucosides.

Erwinia chrysanthemi is one of the few members of the family Enterobacteriaceae that is capable of metabolizing most of the naturally occurring beta-glucosides. We previously isolated the clb genes, which allow the use of the disaccharide cellobiose as well as the aromatic beta-glucosides arbutin and salicin. We report here the isolation of the arb genes, which permit fermentation of the aromatic beta-glucosides only. Establishment of a functional Arb system in Escherichia coli depended on the presence of the phosphotransferase system and on the activation by the cyclic AMP-cyclic AMP receptor protein complex. Strains carrying mini-Mu-induced LacZ fusions to the arb genes were used to analyze arb genes organization and function. Three arb genes (arbG, arbF, and arbB) were identified and organized in this order. Genetic and structural evidence allowed us to assign a phospho-beta-glucosidase and a permease activity to the ArbB and ArbF proteins, respectively. Several Lac+ arb-lacZ insertions were introduced into the E. chrysanthemi chromosome. Both ArbG- and ArbF- strains were unable to ferment the aromatic beta-glucosides, whereas ArbB- strains were impaired only in salicin fermentation. None of the mutations in the arb genes affected cellobiose metabolism. The expression of the arb genes was substrate inducible and required the ArbF permease and, possibly, the ArbG protein. Collectively, our results underline the resemblance between the naturally expressed E. chrysanthemi arbGFB and the cryptic E. coli bglGFB operons, yet the arbG gene product seemed unable to activate E. coli bgl operon expression.     

Molecular cloning, nucleotide sequence, and marker exchange mutagenesis of the exo-poly-alpha-D-galacturonosidase-encoding pehX gene of Erwinia chrysanthemi EC16.

The pehX gene encoding extracellular exo-poly-alpha-D-galacturonosidase (exoPG; EC 3.2.1.82) was isolated from a genomic library of the pectate lyase-deficient Erwinia chrysanthemi mutant UM1005 (a Nalr Kanr delta pelABCE derivative of EC16) by immunoscreening 2,800 Escherichia coli HB101 transformants with an antibody against exoPG protein. The cloned pehX gene was expressed highly from its own promoter in E. coli, and most of the enzyme was localized in the periplasm. The nucleotide sequence of pehX revealed the presence of an amino-terminal signal peptide and an open reading frame encoding a preprotein of 64,608 daltons. The cloned pehX gene was insertionally inactivated with TnphoA and used to mutate the chromosomal pehX gene of E. chrysanthemi AC4150 (Nalr) and CUCPB5006 (Nalr Kans delta pelABCE) by marker exchange mutagenesis. Analysis of the resulting mutants, CUCPB5008 (Pel+ Peh-) and CUCPB5009 (Pel- Peh-), indicated that exoPG can contribute significantly to bacterial utilization of polygalacturonate and the induction of pectate lyase in the presence of extracellular pectic polymers. CUCPB5009 retained a slight ability to pit polygalacturonate semisolid agar and macerated chrysanthemum pith tissues when large numbers of bacteria were inoculated.     

Molecular cloning in Escherichia coli of Erwinia chrysanthemi genes encoding multiple forms of pectate lyase.

The phytopathogenic enterobacterium Erwinia chrysanthemi excretes multiple isozymes of the plant tissue-disintegrating enzyme, pectate lyase (PL). Genes encoding PL were cloned from E. chrysanthemi CUCPB 1237 into Escherichia coli HB101 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322 and then screening recombinant transformants for the ability to sink into pectate semisolid agar. Restriction mapping of the cloned DNA in eight pectolytic transformants revealed overlapping portions of a 9.8-kilobase region of the E. chrysanthemi genome. Deletion derivatives of these plasmids were used to localize the pectolytic genotype to a 2.5-kilobase region of the cloned DNA. PL gene expression in E. coli was independent of vector promoters, repressed by glucose, and not induced by galacturonan. PL accumulated largely in the periplasmic space of E. coli. An activity stain used in conjunction with ultrathin-layer isoelectric focusing resolved the PL in E. chrysanthemi culture supernatants and shock fluids of E. coli clones into multiple forms. One isozyme with an apparent pI of 7.8 was produced at a far higher level in E. coli and was common to all of the pectolytic clones. Activity staining of renatured PL in sodium dodecyl sulfate-polyacrylamide gels revealed that this isozyme comigrated with the corresponding isozyme produced by E. chrysanthemi. The PL isozyme profiles produced by different clones and deletion derivative subclones suggest that the cloned region contains at least two PL isozyme structural genes. Pectolytic E. coli clones possessed a limited ability to macerate potato tuber tissues.     

Cloning and characterization of two genes from Bacillus polymyxa expressing beta-glucosidase activity in Escherichia coli

DNA fragments from Bacillus polymyxa which encode beta-glucosidase activity were cloned in Escherichia coli by selection of yellow transformants able to hydrolyze the artificial chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Restriction endonuclease maps and Southern analysis of the cloned fragments showed the existence of two different genes. Expression of either one of these genes allowed growth of E. coli in minimal medium with cellobiose as the only carbon source. One of the two enzymes was found in the periplasm of E. coli, hydrolyzed arylglucosides more actively than cellobiose, and rendered glucose as the only product upon cellobiose hydrolysis. The other enzyme was located in the cytoplasm, was more active toward cellobiose, and hydrolyzed this disaccharide, yielding glucose and another, unidentified compound, probably a phosphorylated sugar.     

Cloning and characterization of two genes from Bacillus polymyxa expressing beta-glucosidase activity in Escherichia coli.

DNA fragments from Bacillus polymyxa which encode beta-glucosidase activity were cloned in Escherichia coli by selection of yellow transformants able to hydrolyze the artificial chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Restriction endonuclease maps and Southern analysis of the cloned fragments showed the existence of two different genes. Expression of either one of these genes allowed growth of E. coli in minimal medium with cellobiose as the only carbon source. One of the two enzymes was found in the periplasm of E. coli, hydrolyzed arylglucosides more actively than cellobiose, and rendered glucose as the only product upon cellobiose hydrolysis. The other enzyme was located in the cytoplasm, was more active toward cellobiose, and hydrolyzed this disaccharide, yielding glucose and another, unidentified compound, probably a phosphorylated sugar.     

Cloning and analysis of structural and regulatory pectate lyase genes of Erwinia chrysanthemi ENA49

Erwinia chrysanthemi ENA49 structural and regulatory ptl genes, coding for pectate lyase (Ptl) were cloned in Escherichia coli cells. Phage vector lambda L47.1 and phasmid vector lambda pMYF131 were used for constructing libraries of BamHI and EcoRI fragments, respectively, of Er. chrysanthemi chromosomal DNA. Among the 1,100 hybrid clones containing BamHI Er. chrysanthemi DNA fragments and 11,000 hybrid clones containing EcoRI fragments, six and 45 clones, respectively, were identified as having pectolytic activity. Two different structural genes, designated ptlA and ptlB, have been subcloned on multi-copy plasmids. Genes ptlA and ptlB are located side by side on the chromosome of Er. chrysanthemi and transcribe in the same direction. Each of the genes has its own promoter. Southern-blot hybridization analysis showed that the cloned ptl genes shared practically no homology and each of the genes was represented by a single copy on the Er. chrysanthemi chromosome. Other ptl genes capable of expression in E. coli cells were not found in the gene libraries. Negative regulation of the ptlA gene expression by a cloned gene called ptlR was shown. To screen the gene library for the ptlR gene, a specific genetic system was devised. The genes studied are located within an EcoRI chromosomal DNA fragment of 7.3 kb in the order: ptlA-ptlB-ptlR.     

Isolation and genetic study of Erwinia mutants devoid of common components of the phosphoenolpyruvate-dependent phosphotransferase system

Mutants of bacteria belonging the genus Erwinia (Erwinia chrysanthemi and Erwinia carotovora) with pleiotropic disturbances in the utilization of many substrates were obtained through chemical and transposon mutagenesis. Genetic studies revealed that these mutants had defective ptsI or ptsH genes responsible for the synthesis of common components of the phosphoenolpyruvate-dependent phosphotransferase system, enzyme I and the HPr protein, respectively. The ptsI+ allele in both Erwinia species was cloned in vivo. Mapping of obtained mutations indicated that the ptsI and ptsH genes of E. chrysanthemi do not constitute a linkage group. The ptsI gene is located at 100 min of the chromosomal map, whereas the ptsH gene is located at 175 min. Sequencing of a portion of the E. chrysanthemi ptsI gene showed that a product of the cloned DNA region had up to 68% homology with the N terminus of Escherichia coli enzyme I.     

Cloning of pectate-lyase genes of Erwinia chrysanthemi in Escherichia coli cells

Erwinia chrysanthemi DNA fragment digested by restriction endonuclease EcoRI and carrying the gene EC16 determining the synthesis of pectatelyase with Rf 0.20 and mol. mass 40kD has been cloned in plasmid pUC 9 plasmid in Escherichia coli HB101 cells. Three genes for pectatelyases of Erwinia chrysanthemi ENA49 have been cloned in vector phage lambda 47.1 in Escherichia coli cells. Two genes determining the synthesis of pectatelyases with Rf 0.06 and 0.19 and mol. masses 40 kD and 39 kD have been cloned as a part of an 7 kb Eco RI-fragment, that suggested their close location on the chromosome of Erwinia chrysanthemi ENA49. All of the cloned pectatelyase genes are expressed constitutively with pectatelyases accumulating in periplasm and being unable to secret into the cultural medium.     

Cloning of genes encoding extracellular metalloproteases from Erwinia chrysanthemi EC16.

A 14-kilobase BamHI-EcoRI DNA fragment cloned from Erwinia chrysanthemi EC16 contained a gene encoding a metalloprotease inhibitor as well as three tandem prt genes encoding metalloproteases. The prt genes were separated from the inhibitor gene by a ca. 4-kilobase region that was necessary for extracellular secretion of the proteases. When individually subcloned downstream from vector promoters, the three prt genes each led to substantial extracellular secretion of the proteases by Escherichia coli cells, provided that the 4-kilobase required region was supplied in cis or trans. One of the protease structural genes, prtC, was sequenced and had high homology to a metalloprotease gene previously described from Serratia species as well as to the prtB gene of E. chrysanthemi B374. Marker exchange mutants of E. chrysanthemi EC16 defective in production of one or all of the extracellular proteases were not impaired in virulence on plant tissue.     

Lactose metabolism in Erwinia chrysanthemi.

Wild-type strains of the phytopathogenic enterobacterium Erwinia chrysanthemi are unable to use lactose as a carbon source for growth although they possess a beta-galactosidase activity. Lactose-fermenting derivatives from some wild types, however, can be obtained spontaneously at a frequency of about 5 X 10(-7). All Lac+ derivatives isolated had acquired a constitutive lactose transport system and most contained an inducible beta-galactosidase. The transport system, product of the lmrT gene, mediates uptake of lactose in the Lac+ derivatives and also appears to be able to mediate uptake of melibiose, raffinose, and galactose. Two genes encoding beta-galactosidase enzymes were detected in E. chrysanthemi strains. That mainly expressed in the wild-type strains was the lacZ product. The other, the lacB product, is very weakly expressed in these strains. These enzymes showed different affinities for the substrates o-nitrophenyl-beta-D-galactopyranoside and lactose and for the inhibitors isopropyl-beta-D-thiogalactopyranoside and galactose. The lmrT and lacZ genes of E. chrysanthemi, together with the lacI gene coding for the regulatory protein controlling lacZ expression, were cloned by using an RP4::miniMu vector. When these plasmids were transferred into Lac- Escherichia coli strains, their expression was similar to that in E. chrysanthemi. The cloning of the lmrT gene alone suggested that the lacZ or lacB gene is not linked to the lmrT gene on the E. chrysanthemi chromosome. One Lac+ E. chrysanthemi derivative showed a constitutive synthesis of the beta-galactosidase encoded by the lacB gene. This mutation was dominant toward the lacI lacZ cloned genes. Besides these mutations affecting the regulation of the lmrT or lacB gene, the isolation of structural mutants unable to grow on lactose was achieved by mutagenic treatment. These mutants showed no expression of the lactose transport system, the lmrT mutants, or the mainly expressed beta-galactosidase, lacZ mutants. The lacZ mutants retained a very low beta-galactosidase level, due to the lacB product, but this level was low enough to permit use of the lacZ mutants for the construction of gene fusions with the Escherichia coli lac genes.     

Addition of genes for cellobiase and pectinolytic activity in Escherichia coli for fuel ethanol production from pectin-rich lignocellulosic biomass

Ethanologenic Escherichia coli strain KO11 was sequentially engineered to contain the Klebsiella oxytoca cellobiose phosphotransferase genes (casAB) as well as a pectate lyase (pelE) from Erwinia chrysanthemi, yielding strains LY40A (casAB) and JP07 (casAB pelE), respectively. To obtain an effective secretion of PelE, the Sec-dependent pathway out genes from E. chrysanthemi were provided on a cosmid to strain JP07 to construct strain JP07C. Finally, oligogalacturonide lyase (ogl) from E. chrysanthemi was added to produce strain JP08C. E. coli strains LY40A, JP07, JP07C, and JP08C possessed significant cellobiase activity in cell lysates, while only strains JP07C and JP08C demonstrated extracellular pectate lyase activity. Fermentations conducted by using a mixture of pure sugars representative of the composition of sugar beet pulp (SBP) showed that strains LY40A, JP07, JP07C, and JP08C were able to ferment cellobiose, resulting in increased ethanol production from 15 to 45% in comparison to that of KO11. Fermentations with SBP at very low fungal enzyme loads during saccharification revealed significantly higher levels of ethanol production for LY40A, JP07C, and JP08C than for KO11. JP07C ethanol yields were not considerably higher than those of LY40A; however, oligogalacturonide polymerization studies showed an increased breakdown of biomass to small-chain (degree of polymerization, ≤6) oligogalacturonides. JP08C achieved a further breakdown of polygalacturonate to monomeric sugars, resulting in a 164% increase in ethanol yields compared to those of KO11. The addition of commercial pectin methylesterase (PME) further increased JP08C ethanol production compared to that of LY40A by demethylating the pectin for enzymatic attack by pectin-degrading enzymes.     

Role of the nucleoid-associated protein H-NS in the synthesis of virulence factors in the phytopathogenic bacterium Erwinia chrysanthemi

The ability of the enterobacterium Erwinia chrysanthemi to induce pathogenesis in plant tissue is strongly related to the massive production of plant-cell-wall-degrading enzymes (pectinases, cellulases, and proteases). Additional factors, including flagellar proteins and exopolysaccharides (EPS), also are required for the efficient colonization of plants. Production of these virulence factors, particularly pectate lyases, the main virulence determinant, is tightly regulated by environmental conditions. The possible involvement of the protein H-NS in this process was investigated. The E. chrysanthemi hns gene was cloned by complementation of an Escherichia coli hns mutation. Its nucleotide sequence contains a 405-bp open reading frame that codes for a protein with 85% identity to the E. coli H-NS protein. An E. chrysanthemi hns mutant was constructed by reverse genetics. This mutant displays a reduced growth rate and motility but an increased EPS synthesis and sensitivity toward high osmolarity. Furthermore, pectate lyase production is dramatically reduced in this mutant. The hns mutation acts on at least two conditions affecting pectate lyase synthesis: induction of pectate lyase synthesis at low temperatures (25 degrees C) is no longer observed in the hns mutant and induction of pectate lyase production occurs in the late stationary growth phase in the hns background, instead of in the late exponential growth phase as it does in the parental strain. Moreover, the E. chrysanthemi hns mutant displays reduced virulence on plants. Taken together, these data suggest that H-NS plays a crucial role in the expression of the virulence genes and in the pathogenicity of E. chrysanthemi.     

Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

Directed evolution of cellobiose utilization in Escherichia coli K12

The cellobiose catabolic system of Escherichia coli K12 is being used to study the role of cryptic genes in evolution of new functions. Escherichia coli does not use beta-glucoside sugars; however, mutations in several loci can activate the cryptic bgl operon and permit growth on the beta-glucoside sugars arbutin and salicin. Such Bgl+ mutants do not use cellobiose, which is the most common beta-glucoside in nature. We have isolated a Cel+ (cellobiose-utilizing) mutant from a Bgl+ mutant of E. coli K12. The Cel+ mutant grows well on cellobiose, arbutin, and salicin. Genes for utilization of these beta-glucosides are located at 37.8 min on the E. coli map. The genes of the bgl operon are not involved in cellobiose utilization. Introduction of a deletion covering bgl does not affect the ability to utilize cellobiose, arbutin, or salicin, indicating that the new Cel+ genes provide all three functions. Spontaneous cellobiose negative mutants also become arbutin and salicin negative. Analysis of beta-glucoside positive revertants of these mutants indicates that there are separate loci for utilization of each of the beta-glucoside sugars. The genes are closely linked and may be activated from a single locus. A fourth gene at an unknown location increases the growth rate on cellobiose. The cel genes constitute a second cryptic system for beta-glucoside utilization in E. coli K12.      

Functional genes for cellobiose utilization in natural isolates of Escherichia coli.

The genes for utilization of cellobiose are normally cryptic in both laboratory strains and natural isolates of Escherichia coli. A survey of natural isolates of E. coli reveals that functional genes for cellobiose utilization, while rare, are present. The fraction of E. coli that utilized cellobiose ranged from less than 0.01% in human fecal samples to 7% in fecal samples obtained from horses. Samples obtained from sheep, cows, dogs, and pigs contained 0.1 to 0.5% cellobiose-positive E. coli. Neither the previously identified cel genes nor the bgl genes from E. coli K-12 were expressed during growth on cellobiose by any of the 14 naturally occurring Cel+ isolates that were tested. All of the naturally occurring Cel+ isolates possessed a cel operon, but all were deleted for the major portion of the bgl operon. The functional cel+ genes from these natural isolates differed from the mutationally activated cel+ genes obtained in earlier studies in that (i) the mutationally activated cel+ genes were temperature sensitive, while the functional genes were not, and (ii) transport of cellobiose was inducible in the strains carrying functional cel+ genes, while it was expressed constitutively in strains carrying mutationally activated genes.     

Genetic Transfer of Episomic Elements Among Erwinia Species and Other Enterobacteria: F′lac+

The episomic element F'lac(+) was transferred, probably by conjugation, from Escherichia coli to Lac(-) strains of Erwinia herbicola, Erwinia amylovora, and Erwinia chrysanthemi (but not to several other Erwinia spp. In preliminary trials). The lac genes in the exconjugants of the Erwinia spp. showed varying degrees of stability depending on the strain (stable in E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but markedly unstable in E. chrysanthemi strain EC16). The lac genes and the sex factor (F) were eliminated from the exconjugants by treatment with acridine orange, thus suggesting that both lac and F are not integrated in the Erwinia exconjugants. All of the tested Lac(+) exconjugants of E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but not of E. chrysanthemi strain EC 16, were sensitive to the F-specific phage M13. The heterogenotes (which harbored F'lac(+)) of E. herbicola strains Y46 and Y74, E. amylovora strain EA178, and E. chrysanthemi strain EC16 were able to transfer lac genes by conjugation to strains of E. herbicola, E. amylovora, E. chrysanthemi, Escherichia coli, and Shigella dysenteriae. The frequency of such transfer from Lac(+) exconjugants of Erwinia spp. was comparable to that achieved by using E. coli F'lac(+) as donors, thus indicating the stability, expression, and restriction-and-modification properties of the sex factor (F) in Erwinia spp.     

Marker-exchange mutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi.

The phytopathogenic enterobacterium Erwinia chrysanthemi contains pel genes encoding several different isozymes of the plant-tissue-disintegrating enzyme pectate lyase (PL). The pelC gene, encoding an isozyme with an approximate isoelectric point of 8.0, was mutagenized by a three-step procedure involving (i) insertional inactivation of the cloned gene by ligation of a kan-containing BamHI fragment from pUC4K with a partial Sau3A digest of E. chrysanthemi pelC DNA in pBR322; (ii) mobilization of the pBR322 derivative from Escherichia coli to E. chrysanthemi by the helper plasmids R64drd11 and pLVC9; and (iii) exchange recombination of the pelC::kan mutation into the E. chrysanthemi chromosome by selection for kanamycin resistance in transconjugants cultured in phosphate-limited medium (which renders pBR322 unstable). The resulting E. chrysanthemi mutant was Kanr Amps, lacked pBR322 sequences, and was deficient in only one of the four major PL isozymes, PLc, as determined by activity-stained isoelectric-focusing polyacrylamide gels. The rates of PL induction and cell growth in a medium containing polygalacturonic acid as the sole carbon source were not significantly reduced in the mutant. No difference was detected in the ability of the mutant to macerate potato tuber tissue. The evidence suggests that this isozyme is not necessary for soft-rot pathogenesis.     

Secretion of the Rhizobium leguminosarum nodulation protein NodO by haemolysin-type systems

The Rhizobium leguminosarum biovar viciae nodulation protein NodO is partially homologous to haemolysin of Escherichia coli and, like haemolysin, is secreted into the growth medium. The NodO protein can be secreted by a strain of E. coli carrying the cloned nodO gene plus the haemolysin secretion genes hlyBD, in a process that also requires the outer membrane protein encoded by tolC. The related protease secretion genes, prtDEF, from Erwinia chrysanthemi also enable E. coli to secrete NodO. The Rhizobium genes encoding the proteins required for NodO secretion are unlinked to nodO and are unlike other nod genes, since they do not require flavonoids or NodO for their expression. Although proteins similar to NodO were not found in rhizobia other than R. leguminosarum bv. viciae, several rhizobia and an Agrobacterium strain containing the cloned nodO gene were found to have the ability to secrete NodO. These observations indicate that a wide range of the Rhizobiaceae have a protein secretion mechanism analogous to that which secretes haemolysin and related toxins and proteases in the ENterobacteriaceae.