Isolation of Erwinia chrysanthemi mutants altered in pectinolytic enzyme production

Various mutations in the pectin catabolic pathway of Erwinia chrysanthemi were isolated by selection of Mu-lac insertions, resulting in expression of the lac genes inducible by pectin degradation products. This approach allowed us to isolate lacZ fusions with the genes pelC, pelD, ogl and pem, encoding pectate lyases PLc and PLd, oligogalacturonate lyase and pectin methytesterase, respectively. Moreover, we obtained mutations affecting the regulation of pectinolytic enzymes; a locus called peel appeared to be involved in induction of pectate tyases and pectin methylesterase. A second locus, called pect, may encode an activator protein acting on pectate lyase production. Both peel and pecL expression are induced in the presence of pectic polymers. The expression of the pem gene was studied in more detail by analysis of the pem-lacZ fusions. The expression of pem appears to be controlled by the negative regulatory gene kdgR, which controls alt the genes involved in pectin degradation (pem, pel, ogl, kduD, kduf, kdgK, kdgA). This study confirmed that 2-keto-3-deoxy-gluconate is a key intermediate for the induction of the pectin catabolic pathway. The three genes pem, pelD and pecl were localized in the same region, near the ade-377 marker on the genetic map of the E. chrysanthemi strain 3937. The pem gene was located more precisely on an 18kb DNA fragment containing the pelADE cluster. However, this 18 kb DNA fragment did not complement the pecl mutation. The pecL mutations were located near the ile-2 marker on the genetic map of E. chrysanthemi strain 3937.     

The PecM protein is necessary for the DNA-binding capacity of the PecS repressor, one of the regulators of virulence-factor synthesis in Erwinia chrysanthemi

The pecS regulatory locus is responsible for the down-expression of many virulence genes in Erwinia chrysanthemi. This locus consists of two genes, pecS and pecM, divergently transcribed. Genetic evidence indicates that the PecM protein modulates the regulatory activity of PecS. Purification and characterization of PecS, expressed either from E. coli, from the wild-type E. chrysanthemi strain or from a pecM mutant, showed that the PecS protein produced in these three genetic backgrounds displays the same biochemical properties. Band-shift assay analysis with the three PecS isoforms confirmed the involvement of the PecM protein in modulating the PecS DNA-binding capacity. Moreover, determination of the Kd_app for operator regions of the PecS protein, produced either by the wild-type E. chrysanthemi or by E. coli, reveals similar affinities. Thus, in E. coli, there is likely to be at least one other PecM-like protein able to cross-react with the E. chrysanthemi PecS protein.     

Pectate lyase gene regulatory mutants of Erwinia chrysanthemi.

The pelB gene, which encodes one of the five pectate lyase isoenzymes of Erwinia chrysanthemi 3937, was mutagenized with a mini-Mu transposable element that can form gene fusions to the neomycin phosphotransferase-encoding region. Secondary mutants resistant to kanamycin in the absence of polygalacturonate, an inducer of wild-type pectate lyase activities, were selected. Such mutants produced other pectate lyase isoenzymes in the absence of the inducer.     

Purification of the acidic pectate lyase and nucleotide sequence of the corresponding gene (pelA) of Erwinia chrysanthemi strain 3937.

The pelA gene from Erwinia chrysanthemi strain 3937, which encodes the acidic pectate lyase, PLa, has been sequenced and characterized. The structural gene consists of a 1179 bp open reading frame encoding a polypeptide of 41,555 Da, which includes an N-terminal signal peptide. The deduced amino acid sequence shows a protein very similar to some PLs already sequenced. Cloning of the pelA gene behind the lacZ promoter of the vector pTZ19R allowed overexpression of PLa into a derivative of strain 3937 deleted of the other pel genes. The mature protein was obtained in milligram amounts from the supernatant of this strain and at homogeneous purity after two purification steps. Its biochemical properties were similar to those of other PLs. Polyclonal antibodies raised against the purified PLa cross-reacted with the basic pectate lyase PLd, but not with PLe. The role of PLa in pathogenicity is discussed.     

Studies on the Components of Mammalian Urine

Pectate lyase was purified approximately 29-fold to electrophoretic homogeneity from Pseudomonas marginalis N6301. A pectate lyase (PL; EC4.2.2.2) gene of the strain was cloned and expressed in Escherichiacoli. The nucleotides of the PL gene (pel) were sequenced. An open reading frame that encodes a polypeptide (molecular weight: 40,812) composed of 380 amino acids including a 29 amino acid signal peptide was assigned. The structural gene of pel consisted of 1140 base pairs. The nucleotide sequence of the 5′-flanking region of pel showed a consensus sequence of the promoter region of the pectin lyase gene (pnl) in P. marginalis N6301, a Pribnow box, and a ribosome binding site as found in E. coli.     

Factors affecting pectate lyase activity during membrane filtration

Summary This paper presents problems encountered during ultrafiltration of pectate lyase (EC 4.2.2.2) of Erwinia chrysanthemi. Membrane adsorption was related to the chemical nature and charge of the membrane polymer. Simple pre-treatment such as coating with a lysine solution greatly improved recovery efficiency.     

The Three-Dimensional Structure of Aspergillus niger Pectin Lyase B at 1.7-? Resolution

The three-dimensional structure of Aspergillus niger pectin lyase B (PLB) has been determined by crystallographic techniques at a resolution of 1.7 Å. The model, with all 359 amino acids and 339 water molecules, refines to a final crystallographic R factor of 16.5%. The polypeptide backbone folds into a large right-handed cylinder, termed a parallel β helix. Loops of various sizes and conformations protrude from the central helix and probably confer function. The largest loop of 53 residues folds into a small domain consisting of three antiparallel β strands, one turn of an α helix, and one turn of a 3₁₀ helix. By comparison with the structure of Erwinia chrysanthemi pectate lyase C (PelC), the primary sequence alignment between the pectate and pectin lyase subfamilies has been corrected and the active site region for the pectin lyases deduced. The substrate-binding site in PLB is considerably less hydrophilic than the comparable PelC region and consists of an extensive network of highly conserved Trp and His residues. The PLB structure provides an atomic explanation for the lack of a catalytic requirement for Ca²⁺ in the pectin lyase family, in contrast to that found in the pectate lyase enzymes. Surprisingly, however, the PLB site analogous to the Ca²⁺ site in PelC is filled with a positive charge provided by a conserved Arg in the pectin lyases. The significance of the finding with regard to the enzymatic mechanism is discussed.     

Mapping and regulation of the cel genes in Erwinia chrysanthemi

Summary Chromosomal mutations of the celZ and celY genes which encode two different endoglucanases in Erwinia chrysanthemi 3937 were obtained by a three-step procedure: (i) in Escherichia coli, insertions of lacZ fusion-forming mini-Mu bacteriophages in the cel genes cloned on plasmids and screening of cel-lac fusions, (ii) Mu-mediated transduction in E. chrysanthemi of the plasmids carrying the fusions, (iii) recombinational exchange between the plasmidic mutated and the wild-type chromosomal alleles. These mutations allowed mapping of celZ between ura and pan and celY between xyl and met on the linkage map of E. chrysanthemi. The -galactosidase activity of these strains indicated that celZ is expressed in the late exponential and stationary growth phases, while celY expression is almost undetectable.     

Cloning of the<Emphasis Type="Italic"> pelA</Emphasis> gene from<Emphasis Type="Italic"> Bacillus licheniformis</Emphasis> 14A and biochemical characterization of recombinant,thermostable, high-alkaline pectate lyase

The pectate lyase gene pelA from alkaliphilic Bacillus licheniformis strain 14A was cloned and sequenced. The nucleotide sequence corresponded to an open reading frame of 1,026 bp that codes for a 39 amino acid signal peptide and a mature protein with a molecular mass of 33,451 Da. The mature PelA showed significant homology to other pectate lyases belonging to polysaccharide lyase family 1, such as enzymes from different Bacillus spp. and Erwinia chrysanthemi. The pelA gene was expressed in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag, allowing purification to near homogeneity in a one-step procedure. The values for the kinetic parameters K _m and V _max of the fusion protein were 0.56 g/l and 51 µmol/min, respectively. The activity of purified PelA^His was inhibited in the presence of excess substrate. Characterization of product formation revealed unsaturated trigalacturonate as the main product. The yields of unsaturated trigalacturonic acids were further examined for the substrates polygalacturonic acid, citrus pectin and sugar-beet pectin.     

Mu-lac insertion-directed mutagenesis in a pectate lyase gene of Erwinia chrysanthemi.

The pelC gene, which encodes one of the five major pectate lyase (PL) isoenzymes in Erwinia chrysanthemi 3937, designated PLc, was subcloned from a hybrid lambda phage into a pBR322 derivative and mutagenized with a mini-Mu-lacZ transposable element able to form fusions to the lacZ gene. One plasmid (pAD1) which had an inactivated pelC gene and a Lac+ phenotype was selected in Escherichia coli. This plasmid was introduced into Erwinia chrysanthemi, and the pelC::mini-Mu insertion was substituted for the chromosomal allele by homologous recombination. This strain lacks the PLc isoenzyme. This Erwinia chrysanthemi strain has a Lac+ phenotype that is inducible by polygalacturonate, as are the wild-type PL activities.     

Differential effect of dsbA and dsbC mutations on extracellular enzyme secretion in Erwinia chrysanthemi

An Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation has been cloned and sequenced. This gene codes for a periplasmic protein with disulphide isomerase activity that has 69% identity and 94% similarity with the E. coli DsbA protein. An E. chrysanthemi dsbA-uidA fusion mutant has been constructed. dsbA expression seems to be constitutive. This mutant has multiple phenotypes resulting from the absence of disulphide bond formation in periplasmic and secreted proteins. Pectate lyases and the cellulase EGZ are rapidly degraded in the periplasm of the dsbA mutant. E. chrysanthemi synthesizes another periplasmic protein with disulphide isomerase activity, namely DsbC. The dsbC gene introduced on a multicopy plasmid in a dsbA mutant was only partially able to restore EGZ secretion, indicating that even if DsbA and DsbC possess disulphide oxydoreductase activity, they are not completely interchangeable. Moreover, pectate lyases expressed in an E. coli dsbA mutant were very instable but their stability was unaffected in a dsbC mutant. These results indicate that DsbA and DsbC could have different substrate specificities.     

Characterization of a family 3 polysaccharide lyase with broad temperature adaptability, thermo-alkali stability, and ethanol tolerance

The 774-bp pectate lyase gene plyAI4 from Bacillus sp. I4 was cloned and expressed in E. coli. The gene encodes a 257-residue polypeptide (PlyAI4, 28.3 kDa) with the highest identities of 97.3% with a putative pectate lyase from Bacillus subtilis BSn5 (ADV94306) and 60.3% with an identified pectate lyase of the polysaccharide lyase family (PL) 3 from Paenibacillus amylolyticus 27C64 (ADB78774). The purified recombinant PlyAI4 (rPlyAI4) exhibited apparently optimal activity at pH 10.5 ?? 11.0 and 50°C. Compared with the majority of reported alkaline pectate lyases, rPlyAI4 exhibited more residual enzyme activity at 20°C (??45%) or at 70°C (??50%) and better thermostability at 70°C (??60 min half-life at 70°C). In the presence of 20% (v/v) ethanol, pectate lyase activity was enhanced by 0.2 fold. After incubation in 40% (v/v) ethanol at 37°C and pH 8.5 for 1 h, the purified rPelAI4 retained more than 75% of the initial activity. Sequence analysis proposed a new signature block, A-D-G-[V/I]-H, for PL 3 pectate lyases. These properties may prove to be important with regards to PlyAI4 for basic research and industrial application.     

The requirement of chrysobactin dependent iron transport for virulence incited by Erwinia chrysanthemi on Saintpaulia ionantha

To incite a systemic disease on its specific host, Saintpaulia ionantha, the soft-rot Erwinia chrysanthemi strain 3937 requires a functional high affinity iron transport system. Under iron starvation, strain 3937 produces chrysobactin, a novel catechol-type siderophore. Recent advances in the biochemistry and genetics of iron assimilation in E. chrysanthemi are reported. Analysis of leaf intercellular fluid from healthy and infected plants suggests: (i) leaf vessels in which the bacteria develop during infection would be low in free iron and (ii) chrysobactin could be produced in planta.     

Molecular cloning of BPL1, a pectate lyase-like gene in Brassica napus

Pectate lyase (EC 4.2.2.2) is an enzyme involved in the maceration and soft rotting of plant tissue via degradation of cell wall in organisms. Plants as well as bacteria and fungi are capable of producing pectate lyases. Here we report the cloning of a novel full-length cDNA of pectate lyase gene, designated BPL1, from Brassica napus by rapid amplification of cDNA ends. BPL1 cDNA is 1787 bp containing a 1503 bp ORF encoding a 500 amino acid protein precursor. The protein precursor has a potential signal peptide with 22 amino acids. Alignment of sequences shows that there are some extremely conserved amino acids among pectate lyase-like proteins from different plant species, and novel C-terminal domains are found in Arabidopsis and Brassica. Phylogenetic analysis of 50 pectate lyase-like proteins from various species demonstrates the obvious distinction among pectate lyase-like proteins from plants, bacteria and fungi, which are subsequently clustered into three groups. The cloning of BPL1 enables us to explore its diverse roles in higher plants and potential application in crop improvement.     

Stimulation of berberine secretion and growth in cell cultures of Thalictrum minus

Summary An endo-pectate lyase (PL; EC 4.2.2.2), originally cloned fiom the phytopathogenic bacterium Erwinia chrysanthemi EC16, was expressed in recA ^– E. coli strain DK1, purified to a single band by isoelectric focusing and used to induce berberine production in established plant suspension cultures of Thalictrum minus L. subsp. saxatile. Addition of 10^–9M pectate lyase c (PLc) stimulated berberine production and enhanced secretion of the alkaloid into the medium. A lower concentration of PLc, 10^–11M, stimulated a transient two-fold increase in cell growth rate relative to untreated cultures. Parallel changes in L-phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity with the rate of berberine synthesis and the inverse relationship between cell growth and berberine synthesis imply that berberine synthesis is stress-related in this cell line.     

Regulation of the Aspergillus nidulans pectate lyase gene (pelA).

Aspergillus nidulans pectate lyase was purified from culture filtrates. The enzyme catalyzed a random eliminative cleavage reaction, had an apparent molecular weight of 40,000, and a pl of 4.2. Pectate lyase antisera were produced and used to identify pectate lyase clones in a cDNA expression library. Thirteen of 14 clones identified immunologically cross-hybridized. The identity of the single-copy pectate lyase gene, which we designated pelA, was confirmed in two ways. First, several cDNA clones expressed pectate lyase activity in Escherichia coli. Second, targeted mutation of the gene in A. nidulans resulted in complete loss of enzyme activity. pelA encodes a 1,300-nucleotide mRNA that was present in cells grown with polygalacturonic acid as carbon source but absent from cells grown with glucose or acetate as carbon source. Thus, pectate lyase expression is regulated at the level of mRNA accumulation.