Expression of porcine pancreatic phospholipase A2. Generation of active enzyme by sequence-specific cleavage of a hybrid protein from Escherichia coli.

The cDNA coding for the porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in E. coli. Expression of proPLA could only be obtained in the form of intracellular aggregates after fusing the 15 kDa proPLA to a large (greater than or equal to 45 kDa) bacterial peptide. The fusion protein was readily purified from cell lysates, and specifically cleaved. Cleavage of the fusion protein was achieved with either hydroxylamine (at Asn/Gly sequences in the denatured protein), or trypsin (between the pro- and the mature PLA in the renatured fusion protein). The former method releases a proPLA-like enzyme, while the latter directly yields PLA. Renaturation of the fusion protein was made possible by the use of a recently reported new S-sulphonation method. The released (pro)PLA was purified (yields of 2-3 mg/ltr of culture medium), and showed identical properties compared to native (pro)PLA.

Isolation and analysis of genes involved in siderophore biosynthesis in plant-growth-stimulating Pseudomonas putida WCS358.

The plant-growth-stimulating Pseudomonas putida WCS358 was mutagenized with transposon Tn5. The resulting mutant colony bank was screened for mutants defective in the biosynthesis of the fluorescent siderophore. A total of 28 mutants, divided into six different classes, were isolated that were nonfluorescent or defective in iron acquisition or both. These different types of mutants together with the probable overall structure of the siderophore, i.e., a small peptide chain attached to a fluorescing group, suggest a biosynthetic pathway in which the synthesis of the fluorescing group is preceded by the synthesis of the peptide part. A gene colony bank of P. putida WCS358 was constructed with the broad-host-range cosmid vector pLAFR1. This genomic library, established in Escherichia coli, was mobilized into the 28 individual mutants, screening for transconjugants restored in fluorescence or growth under iron-limiting conditions or both. A total of 13 cosmids were found to complement 13 distinct mutants. The complementation analysis revealed that at least five gene clusters, with a minimum of seven genes, are needed for siderophore biosynthesis. Some of these genes seem to be arranged in an operon-like structure.

Exclusion by the IncI plasmid R144 determined by measuring DNA transfer in Escherichia coli conjugations.

Exclusion specified by the IncI plasmid R144 was determined by measuring the amount of donor DNA transferred to appropriate recipient cells. When recipient cells harboured an R144-derived Exc+ recombinant plasmid, the exclusion value determined in that way was comparable with the exclusion value determined by measuring the efficiency of transconjugant colony formation. When recipient cells harboured the plasmid R144drd-3, the exclusion value determined by measuring the amount of donor DNA transferred to recipient cells appeared more valid than the value determined by measuring transconjugant colony formation.

Physical and genetic characterization of the IncI plasmid R144-drd3.

A physical and genetic map of the IncI plasmid R144-drd3 was obtained by determining restriction endonuclease sites and by physical and genetic analysis of cloned fragments, of Tn1 insertion mutants and of deletion mutants.

The pro- and mature forms of the E. coli K-12 outer membrane phospholipase A are identical.

The nucleotide sequence of the pldA gene, coding for the outer membrane (OM) phospholipase A of Escherichia coli K-12, and flanking sequences, was determined. Data were obtained from sequences of overlapping deletions which had been generated in vitro from both ends of the gene, using DNase I in the presence of Mn2+ and Bal31 nuclease. The deduced amino acid sequence of the pldA gene product is the first primary sequence of a membrane-bound phospholipase. The complete PldA protein contains 260 amino acids, which include a putative signal sequence, and has a calculated mol. wt. of 29 946 similar to that of the purified protein. Furthermore we found the N terminus of the purified protein to be blocked and the overall amino acid composition to be consistent with the one deduced from the complete pldA gene. Analysis of proteins synthesized in minicells with a pldA coding plasmid in the presence of 8% ethanol did not reveal any new bands on polyacrylamide gels, whereas the control beta-lactamase clearly showed its unprocessed form under the same conditions. These data are consistent with the empirical prediction from the primary sequence, that the PldA protein lacks any signal peptidase 'target' site. We therefore conclude that the PldA protein is exported to the OM without proteolytic removal of the signal peptide.

Transformability of galE variants derived from uropathogenic Escherichia coli strains.

Transformation of uropathogenic Escherichia coli strains with plasmid DNA was in general unsuccessful or very inefficient. Transformation was much more efficient when galE mutants of such strains, in which the lipopolysaccharide chains appeared shorter, were used as recipients.

Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase.

Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. [3H]thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes.

Exclusion in IncI-type Escherichia coli conjugations: the stage of conjugation at which exclusion operates.

The stage at which exclusion operates in matings between donors belonging to the I-type incompatibility group (IncI) was investigated. Mating between Escherichia coli cells harbouring the I-type plasmid R144 and E. coli cells harbouring the R144-derived recombinant plasmid pRAH308, which causes a hundredfold exclusion, was performed on a membrane filter to test whether mating aggregate formation was disturbed. Besides, level and kinetics of the formation of mating aggregates in mixtures of R144+ donor cells and recipient cells carrying plasmid pRAH308 (exclusion-proficient) was compared with the aggregate formation in mixtures of the donor cells and exclusion-deficient recipient cells. Results from these experiments revealed that the exclusion by pRAH308 does not operate at the level of aggregate formation, but acts at the stage of DNA transfer. The exclusion phenomenon by the recombinant plasmid pRAH308 appeared to be representative for exclusion caused by plasmid R144, since essentially identical results were obtained if plasmid R144 was used as exclusion-determining factor.

Degradation of Escherichia coli chromosomal and plasmid DNA in serum.

Incubation of serum-sensitive [3H]thymidine labelled Escherichia coli PC2166 (RSF1030) and E. coli AM1281 (pBR322) harbouring small plasmids (mol. wt 5.5 X 10(6) and 2.6 X 10(6] in serum resulted in killing of 99.9% of the bacteria within 15 min and in the release of 85% of the radioactivity into the medium after 1 h incubation. The fate of chromosomal and plasmid DNA during incubation of the bacteria in serum was analysed by measurement of the amount of DNA-associated radioactivity, by TCA precipitation, by agarose gel electrophoresis and by the capacity of DNA to transform competent acceptor bacteria. Chromosomal DNA and high molecular weight plasmid DNA were rapidly degraded after 1 h incubation of bacteria in serum. However, low molecular weight plasmid DNA was virtually unaffected and remained physicochemically as well as biologically intact during up to 4 h of incubation of bacteria in serum.

Genetic analysis of complex gene clusters in Escherichia coli: the genetic analysis of F72 fimbrial genes.

Cloning techniques make it possible to accommodate bacterial genes on vector DNA molecules. On that basis the investigation of bacterial structures and functions got new impetus. The potentials of molecular genetics for detailed analysis of bacterial structures are illustrated in this paper for the gene cluster involved in the expression of F72 fimbriae associated with a uropathogenic Escherichia coli O6:K2:H1:F7 strain.

Cloning of an exclusion-determining fragment of the IncI plasmid, R144.

By cloning a distinct 8 MDa fragment of the IncI plasmid, R144, in the vector pACYC184, two recombinant plasmids were isolated. In these plasmids, pRAH303 and pRAH308, the inserted fragment was in opposite orientations. Both plasmids when present in a recipient strain caused a conjugation-specific exclusion in crosses with donor cells carrying the IncI plasmid R144. Some derivatives of the recombinant plasmids in which parts were deleted, or in which Tn5 transposons were inserted, appeared to be exclusion negative. Analysis in minicells of the gene products of such plasmids together with those of the original recombinant plasmids revealed that the presence of two proteins, with apparent molecular weights of 13,000 and 19,000 Da could be correlated with the exclusion phenomenon.

Genetic effects of some platinum co-ordination complexes on E.coli DNA as revealed by transformation studies.

E. coli chromosomal DNA was treated with various Pt co-ordination compounds and then used as donor DNA in E. coli transformation. Genetic analysis of transformants obtained with Pt-treated DNA showed effects of cis-diamminedichloroplatinum(II) (cis-Pt(II)) and cis-Pt-dimethyl-1,3-diaminopropane Cl4 (cis-Pt(IV) (DMDAP)) on the processing of the DNA. With trans-diamminedichloroplatinum(II) (trans-Pt(II)) applied in similar concentrations no effects were found. The effects of cis-Pt(II) and cis-Pt(IV) (DMDAP) on the genetic processing were different. The effects of cis-Pt(II) could be explained by assuming intra-strand crosslinks as an important lesion.

Transformation in Escherichia coli: stages in the process.

Transformation experiments with Escherichia coli recipient cells and linear chromosomal deoxyribonucleic acid (DNA) are reported. E. coli can be rendered competent for DNA uptake by a temperature shock (0 degrees C leads to 42 degrees C leads to 0 degrees C) of the recipient cells in the presence of a high concentration of either Ca2+ or Mg2+ ions. Uptake of DNA into a deoxyribonuclease-resistant form, for which the presence of Ca2+ is essential, was possible during the temperature shock but appeared to occur most readily after the heat shock during incubation at 0 degrees C. When DNA was added to cells that had been heat shocked in the presence of divalent cations only, DNA uptake also occurred. This suggests that competence induction and uptake may be regarded as separate stages. Under conditions used to induce competence, we observed an extensive release of periplasmic enzymes, probably reflecting membrane damage induced during development of competence. After the conversion of donor DNA into a deoxyribonuclease-resistant form, transformants could be selected. It appeared that incubation, before plating, of the transformation mixture in a medium containing high Ca2+ and Mg2+ concentrations and supplemented with all growth requirements increased the transformation frequency. This incubation probably causes recovery of physiologically labile cells.

Role of recBC nuclease in Escherichia coli transformation.

In Escherichia coli transformation with linear donor deoxyribonucleic acid, the recBC pathway is functional, but genetic analysis shows that the recBC nuclease is deleterious to linear deoxyribonucleic acid.

On the role of the recipient cell during conjugation in Escherichia coli.

To study the role of the E. coli recipient cell in conjugation recipient cell mutants deficient in conjugation (Con-) were isolated. Mutants specific for F-type E. coli donor cells (ConF-) and mutants specific deficient in conjugation with I-type donor cells (ConI-) were isolated. Both ConF- and ConI- mutants were blocked in stable mating pair formation. Biochemical analysis of the mutants suggests that the outer membrane protein coded by the ompA gene and LPS are important for recipient activity in F-type conjugation while LPS is important for recipient activity in I-type conjugation.

Characterization of the E. coli K12 strain AB1157 as impaired in guanine/xanthine metabolism.

The widely used E. coli K12 strain AB1157 is impaired in guanine (xnathine) metabolism. Mutants blocked in purine biosynthesis before the stage of inosine monophosphate synthesis do not grow on external guanine or xanthine. The genetic nature of the Gua/Xan lesion is a deletion in the chromosome that covers the proA gene. The lesion causes reduced uptake of guanine.

Conjugation deficient E. coli K12 F- mutants with heptose-less lipopolysaccharide.

Two F- mutants deficient in conjugation with F-type donors are isolated and characterized. Phenotypically, these mutants are similar; they have heptose-less lipopolysaccharide and lack some outer membrane protein. Genotypically, they are different. One mutant harbors a point mutation in the 70 to 74 min region, while the other is deleted for the chromosomal region 6.5 to 8.5 min. Comparison of the properties of the conjugation-deficient mutants described in this paper with other such mutants suggests that an outer membrane protein is the receptor for the f-pilus.

Characterization of an Escherichia coli K-12 F-Con-mutant.

An Escherichia coli K-12 F-mutant defective in conjugation was isolated by means of a zygotic induction enrichment procedure. The recipient ability of the mutant was reduced about 50 times owing to a block in one of the first steps of the conjugation process. In the mutant, cell envelope alterations could not be observed. Sensitivity toward detergents, antibiotics, and phages was unaltered. The mutation appeared to be co-transducible with pyrD. The linkage order in the region of the mutation is origin KL 99-con-pyrD-aroA.