[What can modern genetic methods contribute to clarifying the pathogenicity mechanisms of microorganisms?].
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Bacteria↗
Biomedical subjects
W Goebel
Publications and source records attributed to W Goebel.
Expression and regulation of the plasmid-encoded hemolysin determinant of Escherichia coli.
As a first approach towards studying the regulation of hemolysin synthesis in Escherichia coli, we have fused lacZ into the four hly genes (hlyC, hlyA, hlyBa and hlyBb) using the Mud-1 (Mu::lacZ, Y, Apr) phage. The sites of insertion of Mud-1 within the various hly genes of the Hly plasmid pHly152 were determined by the hemolytic phenotype of the Hly- mutants (Hly-ex/Hly-in or Hly-ex/Hly+in) and by complementation of these Hly- mutants with recombinant plasmids carrying cloned hly genes. It was found that hlyC, hlyA and hlyBa are transcribed from a relatively weak promoter (hlypL) located in front of hlyC. The activity of beta-galactosidase is considerably lower when Mud-1 is integrated in hlyBa than when it is inserted in hlyC, suggesting a considerable decline in hly gene expression from hlyC to hlyBa. The DNA sequence upstream of the coding region of hlyC was found to promote galK gene expression when a fragment covering this region was inserted into the promoter-probe vector pKO-11. A putative promoter sequence, which could correspond to hlypL, was identified in this sequence. The hlyBb gene appears to be transcribed from a different promoter and the direction of transcription seems to be opposite to that of the hlyC, A, Ba operon. The strength of this promoter (hlypR), based on the level of beta-galactosidase activity of Mud-1 insertion mutants in hlyBb, is considerably higher than that of hlypL.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Multiple copies of hemolysin genes and associated sequences in the chromosomes of uropathogenic Escherichia coli strains.
The O6 serogroup Escherichia coli strain 536 carries two hemolysin (hly) determinants integrated into the chromosome. The two hly determinants are not completely identical, either functionally or structurally, as demonstrated by spontaneous deletion mutants carrying only one of them and by cloning each of the two determinants separately into cosmid vectors. Each hly determinant is independently deleted at a frequency of 10(-4), leading to variants which exhibit similar levels of internal hemolysin but different amounts of secreted hemolysin. The two hly determinants were also identified in the O4 E. coli strain 519. The three E. coli strains 251, 764, and 768, which belong to the serogroup O18, and the O4 strain 367 harbor a single chromosomal hly determinant, as demonstrated by hybridization with hly-gene-specific probes. However, a hybridization probe derived from a sequence adjacent to the hlyC-proximal end of the plasmid pHly 152-encoded hly determinant hybridizes with several additional chromosomal bands in hemolytic O18 and O6 E. coli strains and even in E. coli K-12. The size of the probe causing the multiple hybridization suggests a 1,500- to 1,800-base pair sequence directly flanking hlyC. Spontaneous hemolysin-negative mutants were isolated from strains 764 and 768, which had lost the entire hly determinant but retained all copies of the hlyC-associated sequence.2+.
Base Sequence↗
Chromosomal mutation that affects excretion of hemolysin in Escherichia coli.
Two types of mutants unable to excrete hemolysin were obtained when E. coli 5K carrying the multicopy hemolytic recombinant plasmid pANN202-312 was mutagenized with Mu d1. One type is altered in the plasmid hly-specific gene, hlyBb, but the other is caused by an insertion of Mu d1 into a chromosomal locus.
Aminocoumarins↗
Study of regulation and transport of hemolysin by using fusion of the beta-galactosidase gene (lacZ) to hemolysin genes.
Operon and gene fusions between lacZ and the hemolysin genes, hlyC and hlyA, were performed. These two genes are essential for the synthesis of active hemolysin and are transcribed from a common promoter (p1). Whereas the amount of hemolysin produced in Escherichia coli is not changed by altering the hly gene dose, beta-galactosidase activity follows the gene dosage in both types of fusions when lacZ comes under the control of p1. This indicates that hemolysin is not negatively regulated on the transcription or translation level. The products of the gene fusions hlyC::lacZ and hlyA::lacZ were identified in maxicells as stable proteins of 146,000 and 220,000 daltons, respectively. Both fusion proteins possess beta-galactosidase activity indicating that the performed fusions of lacZ to the hly genes do not destroy the reading frame of hlyC and hlyA. The fusion proteins HlyC-beta-gal and HlyA-beta-gal were predominantly detected in the cytoplasm, confirming previous data which suggested that the primary gene products of hlyC and hlyA are not transported across the cytoplasmic membrane.
Bacterial Proteins↗
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.
Base Sequence↗
Expression of antibiotic resistance genes from Escherichia coli in Bacillus subtilis.
Bifunctional recombinant plasmids were constructed, comprised of the E. coli vectors pBR322, pBR325 and pACYC184 and different plasmids from Gram-positive bacteria, e.g. pBSU161-1 of B. subtilis and pUB110 and pC221 of S. aureus. The beta-lactamase (bla) gene and the chloramphenicol acetyltransferase (cat) gene from the E. coli plasmids were not transcribed and therefore not expressed in B. subtilis. However, tetracycline resistance from the E. coli plasmids was expressed in B. subtilis. Transcription of the tetracycline resistance gene(s) started in B. subtilis at or near the original E. coli promoter, the sequence of which is almost identical with the sequence recognized by sigma 55 of B. subtilis RNA polymerase.
Acetyltransferases↗
Hemolysin production as a virulence marker in symptomatic and asymptomatic urinary tract infections caused by Escherichia coli.
Potential virulence, as defined by combined levels of adhesion to urinary epithelial cells, serum resistance, and mouse toxicity, was assessed for Escherichia coli strains causing symptomatic and asymptomatic urinary tract infections in relation to the carriage of hemolysin and other suspected virulence determinants. Hemolysin production (Hly), associated with certain O (O4, O6, O18, and O75), K (5), and hemagglutination (VI and VII) antigenic types but not colicin V production (Cva), was evident in 83 and 60% of isolates in groups possessing high potential virulence and in only 11 and 6% of those with low virulence. Strains of particular O-types were not more virulent per se, but among the serotypes, specific combinations of virulence factors appeared decisive, e.g., O18 HAVI B/D/G Hly(+) K5(+/-) and O18 HAIII/IVB/V Hly(-) Cva(+/-) K1(+/-) strains were, respectively, of high and low potential virulence. Isolates with high potential virulence were found to a similar extent in symptomatic and asymptomatic infections.
Colicins↗
Cloned hemolysin genes from Escherichia coli that cause urinary tract infection determine different levels of toxicity in mice.
After intraperitoneal injection of mice with Escherichia coli strains isolated from patients with urinary tract infections, the mortality due to hemolytic (Hly+) and nonhemolytic (Hly-) isolates was 77 and 40%, respectively. Deletion of the chromosomal hemolysin (hly) determinant in an E. coli O6:K15:H31 urinary tract infection strain led to a significant reduction in toxicity for mice, and its reintroduction on a recombinant plasmid partially restored the original toxicity. Although introduction of the cloned plasmid pHly152-encoded hly determinant into the Hly- E. coli O6 mutant strain increased toxicity by only a marginal degree, transformation with the cloned chromosomal hly determinants from two E. coli strains of serotypes O18ac:K5:H- and O75:K95:H? resulted in markedly greater toxicity, even exceeding that of the original Hly+ E. coli O6 wild-type strain.
Animals↗
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.
Base Sequence↗
Transport of hemolysin across the outer membrane of Escherichia coli requires two functions.
Among a large collection of hemolysis-negative mutants obtained by mutagenesis of the Hly plasmid pHly152 with Tn5, we have isolated two classes of mutants which are defective in the transport of hemolysin across the outer membrane. The two cistrons (hylBa and hlyBb) which are affected in these mutants are located adjacent to each other on the hly determinant but are transcribed from different promoters. Recombinant plasmids were constructed which carry the two functions as combined or separated cistrons. These were shown to complement the two types of transport mutants. Studies on the compartmentation of hemolysin in these two classes of mutants indicate that most hemolysin (greater than 70%) in hlyBa mutants is located in the periplasmic space, whereas in hlyBb mutants a larger portion of hemolysin is associated with the outer membrane fraction. The phenotypic appearance of colonies from hlyBb mutants is that of beta-hemolytic Escherichia coli strains, indicating that a substantial portion of hemolysin has already reached the outside of the outer membrane without being released into the medium. Release was achieved readily when hlyBb mutants were complemented with a recombinant plasmid carrying hlyBb.
Cell Membrane↗
Spontaneous deletions and flanking regions of the chromosomally inherited hemolysin determinant of an Escherichia coli O6 strain.
The hemolytic Escherichia coli strain 536 (O6) propagates spontaneous hemolysin-negative mutants at relatively high rates (10(-3) to 10(-4)). One type of mutant (type I) lacks both secreted (external) and periplasmic (internal) hemolysin activity (Hlyex-/Hlyin-) and in addition shows no mannose-resistant hemagglutination (Mrh-), whereas the other type (type II) is Hlyex-/Hlyin+ and Mrh+. The genetic determinants for hemolysin production (hly) and for mannose-resistant hemagglutination (mrh) of this strain are located on the chromosome. Hybridization experiments with DNA probes specific for various parts of the hly determinant reveal that mutants of type I have lost the total hly determinant, whereas those of type II lack only part of the hlyB that is essential for transport of hemolysin across the outer membrane. Using a probe that contains the end sequence of the plasmid pHly152-encoded hly determinant (adjacent to hlyB), we determined that a related sequence flanks also the hlyB-distal end of the chromosomal hly determinant of E. coli 536. In addition several other similar or even identical sequences are found in the vicinity of the hlyC- and the hlyB-distal ends of both the chromosomal and the plasmid hly determinants.
Base Sequence↗
Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus.
From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertion was recloned as a PstI fragment into pJKK3-1, a shuttle vector which replicates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no external or internal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at levels comparable to those of the B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin O-producing strain of Streptococcus pyogenes or from listeriolysin-producing strains of Listeria monocytogenes, no positive hybridization signals were obtained. These data suggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.
Bacillus cereus↗
Hemolytic Escherichia coli strains in the human fecal flora as potential urinary pathogens.
Hemolysin production is presumed to be a virulence factor in extraintestinal, e.g. urinary tract, infections caused by Escherichia coli. In order to investigate its incidence among the fecal flora, particularly in combination with other presumptive virulence factors, 369 and 373 fecal isolates from Würzburg, FRG and Lima, Peru respectively were examined. 12% of the former and 4% of the latter were hemolytic (Hly+) compared to 33% of 249 E. coli strains isolated from urinary tract infections in Würzburg. In comparison to those which were non hemolytic (Hly-), Hly+ fecal E. coli isolates were associated to a far greater extent with other factors implicated in urinary tract virulence. 41% of these Hly+ strains (cf. 8% Hly-) possessed mannose resistant hemagglutination (MRHA types V, VI, VII). In addition they belonged to the "common O serogroups" O1, O2, O4, O6, O7, O8, O18, O25, O75 in 61% of cases (cf. 25% Hly-) and they possessed K5 antigen in 13% of instances (cf. 2.5% Hly-). The occurrence of these three virulence factors among Hly+ fecal E. coli strains is very similar to that observed among E. coli urinary isolates. One may conclude that these Hly+ fecal strains constitute a "pool" of potential urinary tract pathogens.
Adult↗
Genetics and pathogenic role of Escherichia coli haemolysin.
While clear evidence exists for the direct involvement of cytolysins in the pathogenesis of Gram-positive bacteria, the significance of Gram-negative haemolysins remains unclear. This paper presents briefly data indicating a role for haemolysin production in infections caused by Escherichia coli and also experiments which have allowed an analysis of the molecular basis of the haemolysis among pathogenic and non-pathogenic strains of this species.
DNA Restriction Enzymes↗
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.
Biological Transport↗
Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli.
We have cloned the chromosomal hemolysin determinants from Escherichia coli strains belonging to the four O-serotypes O4, O6, O18, and O75. The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74. The hemolytic cosmid clones were relatively stable. The inserts were further subcloned either as SalI fragments in pACYC184 or as BamHI-SalI fragments in a recombinant plasmid (pANN202) containing cistron C (hlyC) of the plasmid-encoded hemolysin determinant. Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure. These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin. In the gene banks of two of these hemolytic strains, we could also identify clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc. Both of these fimbrial antigens were expressed in the E. coli K-12 clones to an extent similar to that observed in the wild-type strains. These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency.
Base Sequence↗
Copy number control and incompatibility of plasmid R1: identification of a protein that seems to be involved in both processes.
Investigations into the genetic determinants for incompatibility of miniplasmids and hybrid replicons constructed from wide type and mutant R1 revealed the presence of an incompatibility function at the junction f two small PstI fragments. These two fragments were not distinguished in earlier experiments since they have the same mobility on agarose gels. This incompatibility function is distinct from other inc-determinants of R1 (Kollek and Goebel 1979; Molin and Nordström, 1980) and independent of R1-type replication. By means of specific deletions and subcloning of DNA fragments, the location of this new inc-determinant could be determined further. After deletion of this inc-determinant from inc-determinant from miniplasmids, a 5-fold increase in copy number was observed which could then be reduced to a copy number of about 1 plasmid per cell by complementation with hybrid plasmids having this function. Incompatibility of miniplasmids deleted in this determinant is not reduced, whereas analogous deletions introduced into recombinant plasmids nearly abolished their incompatibility. This determinant seems to exert strong incompatibility only when cloned on pBR322. Therefore, its main function is plasmid R1 is probably restricted to copy control. The appearance of low copy numbers of of miniplasmids carrying this determinant and of trans-acting copy control and strong incompatibility exerted by hybrid plasmids is consistently correlated with the presence of a protein of 11,000 molecular weight, synthesized in relatively large amounts in Escherichia coli minicells.
Bacterial Proteins↗