Relationship between plasmid and chromosomal hemolysin determinants of Escherichia coli

Plasmid hemolysin (hly) determinants have been shown previously to comprise three cistrons (hlyA, hlyB, hlyC), coding for the synthesis and transport of hemolysin. Using recombinant plasmids as specific probes for these cistrons, we were able to analyze the chromosomal hly determinants of nine Escherichia coli strains which belonged to serotypes O4, O6, O18, and O75 and were isolated from urinary tract infections and fecal flora. The chromosomal hly genes shared extensive sequence homology with the cloned plasmid hly determinant. Nevertheless, small differences were observed, and these were found to lie mainly within cistron A (hlyA), which has been shown to determine the hemolysin protein itself. These fine variations were not specific for the O-serotype.     

Cloning and expression of Rhodococcus genes encoding pigment production in Escherichia coli

Pigment was produced by Escherichia coli cells carrying recombinant plasmids pNIL100, pNIL200 and pNIL400 containing DNA from Rhodococcus sp. E. coli cells containing pNIL100 or pNIL200 (with DNA inserts from Rhodococcus sp. JL10 and Rhodococcus sp. ATCC 21145 respectively) produced both blue and pink pigments, while cells containing pNIL400 (with a DNA insert from Rhodococcus sp. ATCC 21145) produced only pink pigment. Colonies of E. coli(pNIL100) and E. coli(pNIL200) were dark blue, whereas E. coli(pNIL400) colonies were pink. No pigment was detected in Streptomyces griseus transformants containing pNIL100, pNIL200 or pNIL400. Restriction endonuclease mapping indicated that the cloned DNA fragments were different. The pigment gene(s) in pNIL200 producing both the blue and pink pigments were contained within a 2.8 kb DNA fragment. The pigments produced by E. coli transformants containing pNIL200 were characterized by visible and UV spectroscopy. No similar pigments were detected in Rhodococcus sp. ATCC 21145.     

Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli.

We cloned the DNA containing the Escherichia coli hemolysin determinant on a small, high-copy plasmid. We generated plasmids containing fragments of this DNA and used them either alone or in two-plasmid complementation systems to define the limits of the structural genes. This system also allowed us to partially characterize the function of each of the gene products in the production and transport of hemolysin. Taken with previously published data, the present experiments indicate the following. (i) At least three cistrons, hlyC, hlyA, and hlyB (these were previously designated cisC, etc. [Noegel et al., Mol. Gen. Genet. 175:343-350, 1979]), contain the specific genetic information for the hemolytic phenotype, (ii) hlyA encodes a 107,000-kilodalton protein, which seems to be an inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity require only the products of hlyA and hlyC. This activity was found in the periplasm; very little hemolysin activity was found in the cytoplasm, suggesting that the hlyC product is required for transport or activation of the hlyA product or both. (iv) Active hemolysin remains in the periplasm in the absence of hlyB function, hence the hlyB product seems to be necessary for the transport of hemolysin to the exterior of the cell. We further show that overproduction of the hlyA product is lethal, probably causing lysis of the cell.     

Characterization of a chromosomal mutant that blocks hemolysin excretion in Escherichia coli

Abstract Alanine dipeptides normally penetrate into Pseudomonas aeruginosa by the way of two transport systems. In peptidase N-deficient mutants, dialanine is unable to use its low affinity transport system. Uptake competition showed that this system harboured a permease common to the transport of the amino acid alanine. This permease permits the penetration of both alanine and alanyl peptides uniquely in the presence of active peptidase N. The uptake of trialanine is independent of the presence of active peptidase N inside bacteria despite the fact that hydrolysis of this tripeptide absolutely requires this activity.     

Cloning, and expression in Escherichia coli K-12, of the chromosomal hemolysin (phospholipase C) determinant of Pseudomonas aeruginosa.

A hemolysin determinant was cloned from Pseudomonas aeruginosa PA103 by inserting Sau3a-generated DNA fragments between the BamHI sites of the lambda replacement vector WL47.1. A 9.5-kilobase HindIII fragment encoding the hemolysin was subcloned from this phage and inserted into the plasmid vector pHC79 to generate the recombinant plasmid pKC95. Escherichia coli K-12 strains harboring pKC95 exhibited zones of hemolysis after several days of growth on blood agar plates. Hemolysis was shown to be due to phospholipase C activity by using the chromogenic substrate p-nitrophenylphosphorylcholine. Deletion mutants of pKC95 were isolated, and polypeptides expressed from these plasmids were examined by using the E. coli minicell system. A polypeptide of 78,000 daltons was associated with phospholipase C activity. The hemolytic activity was cell associated when expressed in E. coli.     

Cloning and orientation of the gene encoding polynucleotide phosphorylase in Escherichia coli.

Mutations which affect the activity of polynucleotide phosphorylase (PNPase) map near 69 min on the bacterial chromosome. This region of the chromosome has been cloned by inserting the kanamycin-resistant transposon Tn5 near the argG and mtr loci at 68.5 min. Large SalI fragments of chromosomal DNA containing the Tn5 element were inserted into pBR322, and selection was made for kanamycin-resistant recombinant plasmids. Two of these plasmids were found to produce high levels of PNPase activity in both wild-type and host strains lacking PNPase activity. The pnp gene was further localized and subcloned on a 4.8 kilobase HindIII-EcoRI fragment. This fragment was shown to encode an 84,000-molecular weight protein which comigrated with purified PNPase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The orientation of the pnp gene was determined by insertion of Tn5 into the 4.8 kilobase fragment cloned in pBR322. Some of the insertions had lost the ability to elevate the level of PNPase activity in the host bacterium. Restriction mapping of the positions of the Tn5 insertions and analysis of plasmid-encoded polypeptides in UV-irradiated maxi-cells indicated that the pnp gene is oriented in the counterclockwise direction on the bacterial chromosome.     

Genetics of hemolysin of Escherichia coli.

The expression of alpha-hemolysin is a property frequently associated with Escherichia coli extraintestinal infections. We have examined the genetic basis for hemolysin expression by an E. coli strain isolated from a human urinary tract infection. The genes necessary for hemolysin synthesis were found to be chromosomal and to map near the ilv gene cluster. Isogenic hly+ and hly derivatives were also prepared and tested for virulence in the chicken embryo model system. Hemolysin was found to be necessary but not in itself sufficient for E. coli virulence in this in vitro model.     

Cloning and the expression of the hemolysin gene of Leptospira pomona pomona in Escherichia coli

The library of Leptospira pomona genes was obtained on phage vector AL 47.1. From this library a recombinant phage carrying the hemolysin gene was selected. The DNA fragment (7.7 kb) of this phage containing the hemolysin gene was subcloned on plasmid pUC19. E. coli clones with hybrid plasmid pDR7 were shown to be hemolytic, but the secretion of hemolysin by E. coli into the culture medium was not observed.     

Synthesis and secretion of hemolysin by Escherichia coli.

Hemolytic Escherichia coli cells were found to synthesize and secrete significant amounts of hemolysin into a mineral salt-glucose medium containing hemoglobin. The release of de novo-synthesized hemolysin was stopped in the presence of energy metabolism inhibitors such as 2,4-dinitrophenol, sodium azide, or potassium cyanide, resulting in an accumulation of intracellular hemolysin. A similar effect was observed in the presence of procaine, a neuroactive drug which inhibits the processing of exoproteins. Small amounts of hemolysin were secreted into the medium within approximately 10 min of inhibition of protein synthesis by chloramphenicol. This represented the final release of preformed periplasmic hemolysin en route to secretion through the outer membrane and was not caused by adsorption of external hemolysin to the cell surface. This secretion was not energy dependent but was inhibited above pH 8 and at low temperatures (10 to 20 degrees C). We concluded that two transport processes are involved in hemolysin secretion. De novo-synthesized hemolysin is extruded by an energy-dependent process through the cytoplasmic membrane and probably requires processing. In the periplasmic space a small internal pool of preformed hemolysin is accumulated temporarily before being transported through the outer membrane. Release of hemolysin through the outer membrane does not require energy or de novo protein synthesis.     

Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase.

Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.     

Antibiotic susceptibility, hemolysin production and haemagglutinating activity of uropathogenic Escherichia coli

The hemolysin production, haemagglutinating activity (HA) with human 0 group erythrocytes and antibiotic susceptibility of 130 uropathogenic Escherichia coli strains were studied. 43% of the strains produced hemolysins and 39% showed haemagglutinating activity. In 12% of the haemagglutinating strains HA was inhibited by D-mannose. 45% of the hemolytic strains showed haemagglutinating activity. There was a significant relationship between hemolysin production and haemagglutination activity (p less than 0.05). 85% of the 130 Escherichia coli strains were found to be multiple resistant to antibiotics.     

Cloning of the Escherichia coli recJ chromosomal region and identification of its encoded proteins.

A 9.6-kilobase BamHI-SalI fragment carrying recJ+ was cloned into vector pBR322. Deletion and transposon mutagenesis were used to map the recJ gene on this fragment. The maxicell protein-labeling technique was used to correlate a functional recJ gene with the presence of a polypeptide of 53,000 apparent molecular weight. Two additional genes, one encoding two proteins of 26,000 and 25,000 Mr and the other encoding a 31,000-Mr protein, were mapped on a 3.7-kilobase HindIII-SalI subfragment with recJ. Functions for these adjacent genes are not known; however, insertion mutations in these genes lessen the expression of the putative recJ protein detected in maxicells. A 9.6-kilobase BamHI-SalI fragment carrying the temperature-sensitive mutation recJ147 was also cloned and used for complementation studies to identify other recJ mutations.     

Transport of hemolysin by Escherichia coli

The hemolytic phenotype in Escherichia coli is determined by four genes. Two (hlyC and hlyA) determine the synthesis of a hemolytically active protein which is transported across the cytoplasmic membrane. The other two genes (hlyBa and hlyBb) encode two proteins which are located in the outer membrane and seem to form a specific transport system for hemolysin across the outer membrane. The primary product of gene hlyA is a protein (protein A) of 106,000 daltons which is nonhemolytic and which is not transported. No signal peptide can be recognized at its N-terminus. In the presence of the hlyC gene product (protein C), the 106,000-dalton protein is processed to the major proteolytic product of 58,000 daltons, which is hemolytically active and is transported across the cytoplasmic membrane. Several other proteolytic fragments of the 106,000-dalton protein are also generated. During the transport of the 58,000-dalton fragment (and possible other proteolytic fragments of hlyA gene product), the C protein remains in the cytoplasm. In the absence of hlyBa and hlyBb the entire hemolytic activity (mainly associated with the 58,000-dalton protein) is located in the periplasm: Studies on the location of hemolysin in hlyBa and hlyBb mutants suggest that the gene product of hlyBa (protein Ba) binds hemolysin and leads it through the outer membrane whereas the gene product of hlyBb (protein Bb) releases hemolysin from the outer membrane. This transport system is specific for E coli hemolysin. Other periplasmic enzymes of E coli and heterologous hemolysin (cereolysin) are not transported.     

Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12.

Escherichia coli K-12 gene ppa encoding inorganic pyrophosphatase (PPase) was cloned and sequenced. The 5' end of the ppa mRNA was identified by primer extension mapping. A typical E. coli sigma 70 promoter was identified immediately upstream of the mRNA 5' end. The structural gene of ppa contains 528 base pairs, from which a 175-amino-acid translation product, Mr 19,572, was deduced. The deduced amino acid composition perfectly fitted with that of PPase as previously determined (P. Burton, D. C. Hall, and J. Josse, J. Biol. Chem. 245:4346-4351, 1970). Furthermore, the partial amino acid sequence (residues 1 to 108) of E. coli PPase determined by S. A. Cohen (Ph.D. thesis, University of Chicago, 1978) was the same as that deduced from the nucleotide sequence. This is the first report of the cloning of a PPase gene.