Characterization of the yellow-pigment genes of Erwinia herbicola

A 6.7 kb DNA fragment containing the pigment genes of Erwinia herbicola Eho13 has been cloned into Escherichia coli. These genes were chromosomally encoded in E. herbicola. The entire DNA fragment could be divided into at least three regions. Deletions in Region I resulted in a non-pigmented phenotype, a deletion in Region II resulted in a pink/yellow phenotype, deletions in Region III resulted in either a pink or a non-pigmented phenotype. Tn1000 insertions in the same regions, however, gave different phenotypes. Insertions in Region II produced a pink phenotype. Insertions in Region III resulted in either a light-yellow or a non-pigmented phenotype. Minicell studies showed that the 6.7 kb DNA fragment encoded at least five proteins (50 kDa, 42 kDa, 36 kDa, 35 kDa and 34 kDa). A 2.7 kb HindIII deletion in Region I caused the disappearance of these proteins, suggesting that this 2.7 kb fragment may play a regulatory role in pigment synthesis. Our results also showed that a 4.1 kb EcoRV fragment consisted of Region I and a part of Region II complemented a pink/yellow clone of Eho10 (pHL545), suggesting that the pigments of Eho13 and Eho10 were probably similar or identical.     

Cloning,sequencing and partial characterisation of sorbitol transporter (srlT) gene encoding phosphotransferase system,glucitol/sorbitol-specific IIBC components of Erwinia herbicola ATCC 21998 – Cloning,sequencing and partial characterisation of sorbitol specific transporter (srlT) gene of Erwinia herbicola ATCC 21998

A DNA fragment of approximately 1500 bp, harbouring the sorbitol transport gene (srlT), was amplified from the chromosomal DNA of Erwinia herbicola ATCC 21998 by PCR and cloned in Escherichia coli JM109. Degenerate oligonucleotide primers used were designed based on the conserved regions in the gene sequences within the gut operon of E. coli (Gene Bank accession no. J02708) and the srl operon of Erwinia amylovora (Gene Bank accession no. Y14603). The cloned DNA fragment was sequenced and found to contain an open reading frame of 1473 nucleotides coding for a protein of 491 amino acids, corresponding to a mass of 52410 Da. The nucleotide sequence of this ORF was highly homologous to that of the gutA gene of Escherichia coli gut operon, the srlE gene of Shigella flexrni and the sorbitol transporter gene sequence of Escherichia coli K12 (Gene Bank accession nos. J02708, AE016987 and D90892 respectively). The protein sequence showed significant homology to that of the phosphotransferase system i.e. the glucitol/sorbitol-specific IIBC components of Escherichia coli and Erwinia amylovora (P56580, O32522). The cloned DNA fragment was introduced into a pRA90 vector and the recombinant was used for developing srlT mutants of Erwinia herbicola, by homologous recombination. Mutants obtained were unable to grow on minimal medium with sorbitol. The insertion of the pRA90 vector inside the srlT gene sequence of the mutants was confirmed by DNA-DNA hybridisation.     

Mapping of a carotenogenic gene cluster from Erwinia herbicola and functional identification of six genes

Six genes have been mapped and identified by hybridization or by carotenoid analysis of deletion mutants on a 9 kb chromosomal fragment originating from Erwinia herbicola Eho 10. These genes include crtB, E and I which have been formerly described for Rhodobacter, and three new ones: crtZ encoding lycopene cyclase, crtH for beta-carotene hydroxylase, and crtG for zeaxanthin glycosilase. Their arrangement on the plasmid has been elucidated.     

Identification of carotenoids in Erwinia herbicola and in a transformed Escherichia coli strain

The yellow pigments of Erwinia herbicola Eho 10 and of a transformed Escherichia coli LE392 pPL376 have been identified as carotenoids. HPLC separation, spectra and in some cases mass spectroscopy demonstrated the presence of phytoene (15-cis isomer), beta-carotene (all-trans, 9-cis and 15-cis), beta-cryptoxanthin ( = 3-hydroxy beta-carotene), zeaxanthin (3,3'-dihydroxy beta-carotene) and corresponding carotene glycosides. In addition, lycopene and gamma-carotene accumulated in the presence of the inhibitor 2-(4-chlorophenylthio)-triethylamine.HCl. Carotenoid content in the transformed E. coli was two-fold higher than in E. herbicola. The pattern of the carotenoids was similar in the two organisms. Inactivation of the katF gene in E. coli resulted in an 85% lowering of carotenoid formation, as did the addition of 0.5% glucose to the medium. Suppression of carotenoid formation by inactivation of the katF gene lowered, but did not abolish, the protection offered by carotenoids against inactivation by alpha-terthienyl plus near-ultraviolet light (320-400 nm).     

Cloning and nucleotide sequence of the bglA gene from Erwinia herbicola and expression of β-glucosidase activity in Escherichia coli

Abstract Genomic DNA fragments encoding β-glucosidase activity from the wild-type strain WD4 of Erwinia herbicola were cloned into Escherichia coli . Two clones containing a common fragment encoded a polypeptide of 58000 Da. Cloned β-glucosidase, expressed in E. coli , showed activity against natural β-glucoside sugars except for cellobiose. An open reading frame of 1442 bp termed bglA was identified by nucleotide sequencing and it coded for a protein of 480 amino acids ( M _r 53896) which showed significant homology with β-glucosidases from glycosyl hydrolase family 1.     

Location and cloning of the ketal pyruvate transferase gene of Xanthomonas campestris.

Genes required for xanthan polysaccharide synthesis (xps) are clustered in a DNA region of 13.5 kb in the chromosome of Xanthomonas campestris. Plasmid pCHC3 containing a 12.4-kb insert of xps genes has been suggested to include a gene involved in the pyruvylation of xanthan gum (N.E. Harding, J.M. Cleary, D.K. Caba?as, I. G. Rosen, and K. S. Kang, J. Bacteriol. 169:2854-2861, 1987). An essential step toward understanding the biosynthesis of xanthan gum and to enable genetic manipulation of xanthan structure is the determination of the biochemical function encoded by the xps genes. On the basis of biochemical characterization of an X. campestris mutant which produces pyruvate-free xanthan gum, complementation studies, and heterologous expression, we have identified the gene coding for the ketal pyruvate transferase (kpt) enzyme. This gene was located on a 1.4-kb BamHI fragment of pCHC3 and cloned in the broad-host-range cloning vector pRK404. An X. campestris kpt mutant was constructed by mini-Mu(Tetr) mutagenesis of the cloned gene and then by recombination of the mutation into the chromosome of the wild-type strain.     

Cloning chromosomal lac genes of Klebsiella pneumoniae

The chromosomal gene for beta-galactosidase from Klebsiella pneumoniae strain T17R1 and associated regulatory genes have been cloned as a 5-kb HindIII fragment in the pBR322 plasmid vector. The beta-galactoside permease gene is not present in a functional form in the 5-kb fragment. The K. pneumoniae genes are expressed in an Escherichia coli host. The synthesis of beta-galactosidase is inducible by isopropyl-beta-D-galactosidase (IPTG) and is sensitive to catabolite repression. There appears to be greater homology between the K. pneumoniae and E. coli structural genes for beta-galactosidase than there is between the respective repressor 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.     

Cloning and expression of plasmid genes encoding resistances to chromate and cobalt in Alcaligenes eutrophus.

Resistances to chromate and cobalt were cloned on a 30-kilobase-pair (kb) DNA region from the large Alcaligenes eutrophus plasmid pMOL28 into the broad-host-range mobilizable cosmid vector pVK102. A restriction nuclease map of the 30-kb region was generated. The resistances expressed from the hybrid plasmids after transfer back into A. eutrophus were inducible and conferred the same degree of resistance as the parent plasmid pMOL28. Resistances were expressed in metal-sensitive Alcaligenes strains and related bacteria but not in Escherichia coli. Resistance to chromate was further localized on a 2.6-kb EcoRI fragment, and resistance to cobalt was localized on an adjoining 8.5-kb PstI-EcoRI fragment. When the 2.6-kb EcoRI fragment was expressed in E. coli under the control of a bacteriophage T7 promoter, three polypeptides with molecular masses of 31,500, 21,000, and 14,500 daltons were visible on autoradiograms. The 31,500- and 21,000-dalton polypeptides were membrane bound; the 14,500-dalton polypeptide was soluble.     

Expression of the 4-chlorobenzoate dehalogenase genes from Pseudomonas sp.-CBS3 in Escherichia coli and identification of the gene translation products.

The genes encoding the 4-chlorobenzoate dehalogenase of Pseudomonas sp. strain CBS3 were, in an earlier study, cloned in Escherichia coli DH1 with the cosmid vector pPSA843 and then mobilized to the 4-chlorobenzoate dehalogenase minus strain Pseudomonas putida KT2440. In this paper we report on the expression of 4-chlorobenzoate dehalogenase in these clones and on the polypeptide composition of the active enzyme. The dehalogenase activity in whole cells suspended in 3.2 mM 4-chlorobenzoate (30 degrees C) was determined to be approximately 27 units (micromoles 4-hydroxybenzoate produced per minute) per 100 g of E. coli-pPSA843 cells and approximately 28 units per 100 g of P. putida-pPSA843 cells. Dehalogenase activity in fresh cellular extracts (pH 7.4, 30 degrees C) prepared from the E. coli and P. putida clones was unstable and at least 20-fold lower than that observed with the whole cells. The polypeptide components of the dehalogenase were identified by selective expression of the cloned dehalogenase genes and analysis of the gene translation products. Analysis of dehalogenase activity in omega insertion mutants and deletion mutants circumscribed the dehalogenase genes to a 4.8-kilobase (4.8 kb) stretch of the 9.5-kb DNA fragment. Selective expression of the dehalogenase genes from a cloned 4.8-kb DNA fragment in a maxicell system revealed a 30-kDa polypeptide as one of the components of the dehalogenase system. Selective expression of the dehalogenase genes using the T7 polymerase promoter system revealed the 30-kDa polypeptide and 57- and 16-kDa polypeptide products. Determination of which of the three polypeptides were translated in deletion mutants provided the relative positions of the encoding genes on a single DNA strand and the direction in which they are transcribed.     

Identity of Gram Negative, Yellow Pigmented, Fermentative Bacteria isolated from Plants and Animals

S ummary . Morphological, cultural and biochemical properties of 35 isolates of rod shaped Gram negative, yellow pigmented anaerogenic, fermentative bacteria were compared. The isolates comprised named cultures of Bacterium herbicola, Erwinia lathyri, E. ananas, E. milletiae, E. uredovora and B. typhi flavum , as well as organisms isolated from deer and man which were considered to be related to E. milletiae. The results showed that the organisms were indistinguishable from one another on the basis of the tests employed, and it is concluded that B. herbicola, B. typhi flavum, E. lathyri and E. ananas should be classified as Erwinia herbicola (Düggeli) Dye. Since there was insufficient information regarding the plant pathogenicity of E. milletiae and E. uredovora , it is suggested that they should remain separate species at present. The relationship of this group of organisms to certain coliforms and to flavobacteria is briefly discussed.     

Cloning and expression of bacterial ice nucleation genes in Escherichia coli.

Epiphytic populations of Pseudomonas syringae and Erwinia herbicola are important sources of ice nuclei that incite frost damage in agricultural crop plants. We have cloned and characterized DNA segments carrying the genes (ice) responsible for the ice-nucleating ability of these bacteria. The ice region spanned 3.5 to 4.0 kilobases and was continuous over this region in P. syringae Cit7R1. The cloned fragments imparted ice-nucleating activity in Escherichia coli. Substantial increases in the nucleating activity of both E. coli and P. syringae were obtained by subcloning the DNA fragments on multicopy plasmid vectors. Southern blot analysis showed substantial homology between the ice regions of P. syringae and E. herbicola, although individual restriction sites within the ice regions differed between the two species.     

Cloning and nucleotide sequences of the homoserine dehydrogenase genes (hom) and the threonine synthase genes (thrC) of the gram-negative obligate methylotroph Methylobacillus glycogenes.

We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.     

Identification and cloning of a fur regulatory gene in Yersinia pestis.

Yersinia pestis is one of many microorganisms responding to environmental iron concentrations by regulating the synthesis of proteins and an iron transport system(s). In a number of bacteria, expression of iron uptake systems and other virulence determinants is controlled by the Fur regulatory protein. DNA hybridization analysis revealed that both pigmented and nonpigmented cells of Y. pestis possess a DNA locus homologous to the Escherichia coli fur gene. Introduction of a Fur-regulated beta-galactosidase reporter gene into Y. pestis KIM resulted in iron-responsive beta-galactosidase activity, indicating that Y. pestis KIM expresses a functional Fur regulatory protein. A cloned 1.9-kb ClaI fragment of Y. pestis chromosomal DNA hybridized specifically to the fur gene of E. coli. The coding region of the E. coli fur gene hybridized to a 1.1-kb region at one end of the cloned Y. pestis fragment. The failure of this clone to complement an E. coli fur mutant suggests that the 1.9-kb clone does not contain a functional promoter. Subcloning of this fragment into an inducible expression vector restored Fur regulation in an E. coli fur mutant. In addition, a larger 4.8-kb Y. pestis clone containing the putative promoter region complemented the Fur- phenotype. These results suggest that Y. pestis possesses a functional Fur regulatory protein capable of interacting with the E. coli Fur system. In Y. pestis Fur may regulate the expression of iron transport systems and other virulence factors in response to iron limitation in the environment. Possible candidates for Fur regulation in Y. pestis include genes involved in ferric iron transport as well as hemin, heme/hemopexin, heme/albumin, ferritin, hemoglobin, and hemoglobin/haptoglobin utilization.     

Cloning and expression of two cellulase genes of Clostridium cellulolyticum in Escherichia coli

Two cellulase genes isolated from Clostridium cellulolyticum strain ATCC3519 were cloned in Escherichia coli using plasmid pACYC184. Plasmids pB52 and pB43 were isolated from the transformants producing carboxymethylcellulase (CMCase) and the two cloned CMCase-coding genes were found to be included in two EcoRI fragments of 5.7 kb and 2.6 kb, respectively. These two genes showed no homology. The CMCase-coding genes were found to be contained in a 1.8-kb KpnI-HindIII fragment and a 2.05-kb HindIII-PvuII fragment of the DNA donor strain. Expression of these genes in E. coli was found not to depend on their orientation in the cloning vector. Hybridization experiments between these two fragments and Clostridium thermocellum NCIB10682 DNA fragments carrying genes celA, celB, celC and celD were carried out and some homologies were detected.     

Cloning, characterization, and expression of a gene region from Pseudomonas sp. strain ADP involved in the dechlorination of atrazine.

We previously identified a Pseudomonas sp. strain, ADP, which rapidly metabolized atrazine in liquid culture, agar plates, and soils (R. T. Mandelbaum, D. L. Allan, L. P. Wackett, Appl. Environ. Microbiol. 61:1451-1457, 1995). In this study, we report the cloning and partial characterization of a gene region from Pseudomonas sp. strain ADP that encodes atrazine degradation activity. A 22-kb EcoRI genomic DNA fragment, designated pMD1, was shown to encode atrazine dechlorination activity in Escherichia coli DH5 alpha. Atrazine degradation was demonstrated by a zone-clearing assay on agar medium containing crystalline atrazine and by chromatographic methods. A gene conferring the atrazine-clearing phenotype was subsequently subcloned as a 1.9-kb AvaI fragment in pACYC184, designated pMD4, and was expressed in E. coli. This result and random Tn5 mutagenesis established that the 1.9-kb AvaI fragment was essential for atrazine dechlorination. High-pressure liquid and thin-layer chromatographic analyses were used to rigorously establish that E. coli containing pMD4 degraded atrazine and accumulated hydroxyatrazine. Hydroxyatrazine was detected only transiently in E. coli containing pMD1. This is consistent with the idea that hydroxyatrazine is the first metabolite in atrazine degradation by Pseudomonas sp. strain ADP. A 0.6-kb ApaI-PstI fragment from pMD4, containing the putative atrazine chlorohydrolase gene, hybridized to DNA from atrazine-degrading bacteria isolated in Switzerland and Louisiana.(ABSTRACT TRUNCATED AT 250 WORDS)     

Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity.

Production of a potent urease has been described as a trait common to all Helicobacter pylori so far isolated from humans with gastritis as well as peptic ulceration. The detection of urease activity from genes cloned from H. pylori was made possible by use of a shuttle cosmid vector, allowing replication and movement of cloned DNA sequences in either Escherichia coli or Campylobacter jejuni. With this approach, we cloned a 44-kb portion of H. pylori chromosomal DNA which did not lead to urease activity when introduced into E. coli but permitted, although temporarily, biosynthesis of the urease when transferred by conjugation to C. jejuni. The recombinant cosmid (pILL585) expressing the urease phenotype was mapped and used to subclone an 8.1-kb fragment (pILL590) able to confer the same property to C. jejuni recipient strains. By a series of deletions and subclonings, the urease genes were localized to a 4.2-kb region of DNA and were sequenced by the dideoxy method. Four open reading frames were found, encoding polypeptides with predicted molecular weights of 26,500 (ureA), 61,600 (ureB), 49,200 (ureC), and 15,000 (ureD). The predicted UreA and UreB polypeptides correspond to the two structural subunits of the urease enzyme; they exhibit a high degree of homology with the three structural subunits of Proteus mirabilis (56% exact matches) as well as with the unique structural subunit of jack bean urease (55.5% exact matches). Although the UreD-predicted polypeptide has domains relevant to transmembrane proteins, no precise role could be attributed to this polypeptide or to the UreC polypeptide, which both mapped to a DNA sequence shown to be required to confer urease activity to a C. jejuni recipient strain.     

UV-induced formation of filamentous forms in bacteria of Erwinia genus

Cells of 56 pectolytic Erwinia strains of different origin tested are prone to filamentation after UV-irradiation. The fact makes one possible to consider them natural fil+ organisms. Bacteria E. herbicola (9 strains) that are unable to synthesize pectatelyases are not transformed into filaments after NV-irradiation. The function of fil+ genes is recA-dependent in bacteria E. chrysanthemi ENA49 and is phenotypically analogous to fil+ gene function in E. coli B or lon- mutation in E. coli K12.