Holin locus characterisation from lysogenic Xenorhabdus nematophila and its involvement in Escherichia coli SheA haemolytic phenotype

Lysogeny has previously been described in the entomopathogenic bacteria of the genus Xenorhabdus. Screening of a X. nematophila prophage DNA library on blood agar resulted in the identification of a 5.7-kb locus that caused a haemolytic phenotype when cloned in Escherichia coli, but not in the E. coli sheA null mutant, lacking the SheA cryptic haemolysin. This locus exhibited similarity to lysis genes from lambdoid phages. In particular, it encoded a functional holin able to complement a lambda Sam7 mutant. It is the second time that a locus encoding a functional holin is shown to reveal the SheA haemolytic phenotype in E. coli. The possible role of the holin in extracellular release of SheA is discussed.     

Regulation of haemolysin synthesis in E. coli determined by HLY genes of human origin

Summary We have previously reported the secretion of a 107K polypeptide into the medium from a haemolytic E. coli K12 strain (Mackman and Holland 1984a). In addition, we demonstrated that haemolysin production was correlated with the presence of this polypeptide in the growth medium in a large number of E. coli isolates of human and animal origin (Mackman and Holland 1984b).In this paper we confirm that the 107K polypeptide is indeed haemolysin: both haemolytic activity and the 107K polypeptide show a similar pattern of accumulation during the growth cycle; identical levels are produced in three different growth media; they have the same half-life in minimal medium. The results also show that the expression of haemolysin is not influenced by the growth medium or subject to catabolite repression. However, expression is apparently switched off as cells enter the late exponential phase of growth. Finally, we present data indicating that the previously reported variation in haemolysin production in different media is entirely due to the instability of the haemoolysin itself. Degradation of the 107K polypeptide in the medium was accompanied by the accumulation of a major breakdown product of 60K.     

Characterisation of a Pasteurella multocida esterase gene which confers a hemolytic phenotype in Escherichia coli under anaerobic conditions

Investigation of the hemolytic phenotype under anaerobic growth conditions of an avian Pasteurella multocida strain, PBA100, resulted in the identification and characterisation of a gene encoding an esterase enzyme, mesA, that conferred a hemolytic phenotype in Escherichia coli under anaerobic conditions. MesA appeared to be expressed and functional under anaerobic and aerobic conditions in both E. coli and P. multocida. A P. multocida mesA mutant was generated which resulted in the loss of acetyl esterase activity under anaerobic conditions. However, this mutation did not cause any attenuation of virulence for mice nor a detectable change to the anaerobic hemolytic phenotype of P. multocida. In E. coli MesA appeared to cause hemolysis indirectly by the induction of the latent E. coli K-12 cytolysin, sheA.     

Induction of haemolytic activity in Escherichia coli by the slyA gene product

The Salmonella typhimurium protein SlyA_ST, originally described as a cytolysin, shows sequence similarities to several known bacterial regulatory proteins. A homologue to the slyA_St gene has been localised to min 37 of the Eschericia coli K-12 chromosome and has been designated slyA_EC When introduced in trans on a plasmid, the slyA_EC gene conferred a haemolytic phenotype on wild-type but not clyA-knockout strains of E. coli K-12. The clyA gene encodes a novel haemolysin that is not expressed by wild-type E. coli under tested laboratory conditions. Western and Northern blot analyses, and DNA-band-shift assays support a model whereby the SlyA_EC protein activates clyA expression by binding to the clyA promoter region, thereby supporting the sequence similarity data in suggesting that SlyA_ST is a haemolysin activator rather than being a haemolysin per se.     

Isolation of the Actinobacillus pleuropneumoniae haemolysin gene and the activation and secretion of the prohaemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli

The gene encoding the c. 105 kD secreted haemolysin protein of the porcine pathogen Actinobacillus pleuropneumoniae serotype 1 has been isolated by screening a lambda gt11 expression library in Escherichia coli with antiserum raised against the wild-type protein. A derivative recombinant DNA pJFF702 expressed the hlylA haemolysin gene from the pUC19 lac promoter but the resulting haemolysin I protein remained within the E. coli cell and was haemolytically inactive. Export of the intracellular A. pleuropneumoniae prohaemolysin out into the medium was achieved by the presence in trans of the E. coli haemolysin secretion genes hlyB and hlyD, and high levels of intracellular haemolytic activity were attained similarly by the E. coli post-translational haemolysin activator gene, hlyC. Southern hybridization of A. pleuropneumoniae parental DNA nevertheless indicated only a low degree of nucleotide sequence identity to the haemolysin structural and secretion genes hlyA and hlyB of E. coli. The data show that despite substantial nucleotide sequence divergence the A. pleuropneumoniae serotype 1 haemolysin determinant is closely related to that which is dispersed throughout other Gram-negative human and animal pathogens.     

The hha gene modulates haemolysin expression in Escherichia coli

A mutation in the hha allele results in a large increase in the production of intracellular as well as extracellular haemolysin in Escherichia coli cells harbouring the haemolytic recombinant plasmid pANN202-312. This single gene mutation was located between 490 and 491.6kb on the physical map of the E. coli chromosome. From the DNA sequence of hha a small polypeptide of 8629 Da was predicted and was expressed in minicells. The deduced polypeptide sequence did not show significant similarities to other characterized proteins related to the regulation of gene expression in E. coli, although it was shown that the hha mutation increases cyloplasmic synthesis of haemolysin.     

Secretion of the Rhizobium leguminosarum nodulation protein NodO by haemolysin-type systems

The Rhizobium leguminosarum biovar viciae nodulation protein NodO is partially homologous to haemolysin of Escherichia coli and, like haemolysin, is secreted into the growth medium. The NodO protein can be secreted by a strain of E. coli carrying the cloned nodO gene plus the haemolysin secretion genes hlyBD, in a process that also requires the outer membrane protein encoded by tolC. The related protease secretion genes, prtDEF, from Erwinia chrysanthemi also enable E. coli to secrete NodO. The Rhizobium genes encoding the proteins required for NodO secretion are unlinked to nodO and are unlike other nod genes, since they do not require flavonoids or NodO for their expression. Although proteins similar to NodO were not found in rhizobia other than R. leguminosarum bv. viciae, several rhizobia and an Agrobacterium strain containing the cloned nodO gene were found to have the ability to secrete NodO. These observations indicate that a wide range of the Rhizobiaceae have a protein secretion mechanism analogous to that which secretes haemolysin and related toxins and proteases in the ENterobacteriaceae.     

Chromosomal mutations that increase the production of a plasmid-encoded haemolysin in Escherichia coli

Transposon mutagenesis was used to isolate two Escherichia coli mutants which express very large amounts of haemolysin when carrying the multicopy plasmid pANN202-312. E. coli strain Hha-2 was isolated by Mud1 mutagenesis, and strain Hha-3 by Tn5 mutagenesis. The transposon insertion was chromosomal in both mutants and could be demonstrated to be unrelated to the haemolytic region of the plasmid. The substantial increase in both extracellular and intracellular haemolysin production was dependent upon plasmid copy number and was drastically reduced when either mutant carried the low-copy-number haemolytic plasmid pHly152. In both mutants, the marked increase in extracellular production was dependent upon the specific haemolysin transport genes, hlyB and hlyD. The lack of either gene function resulted in no external haemolysin production. SDS-PAGE analysis showed no change in the pattern of outer-membrane proteins of the mutants, although changes (differing between the two mutants) were seen in their periplasmic proteins. The mutations of both strains (termed hha-2 and hha-3) were mapped at minute 10.5 of the E. coli chromosome. No relation to any known gene affecting gene regulation in E. coli could be found.     

Complementation of mutations in Escherichia coli and Bordetella pertussis by B. pertussis DNA cloned in a broad-host-range cosmid vector

A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24.9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.     

Cloning and analysis of a Borrelia burgdorferi membrane-interactive protein exhibiting haemolytic activity

We cloned the gene encoding a membrane-interactive protein of Borrelia burgdorferi by means of its haemolytic activity in Escherichia coli . The haemolytic activity was erythrocyte-species specific, with progressively decreasing activity for erythrocytes from horse, sheep, and rabbit, respectively. Genetic analysis of the haemolytic determinant revealed two borrelia haemolysin genes, blyA and blyB , that are part of a predicted four-gene operon which is present in multiple copies on the 30 kb circular plasmid(s) of B. burgdorferi B31. blyA encodes a predicted α-helical 7.4 kDa protein with a hydrophobic central region and a positively charged C-terminus, which is structurally homologous to a large group of pore-forming toxins with cytolytic activity. blyB encodes a soluble protein which stabilized BlyA and enhanced haemolytic activity. While the majority of BlyA in E. coli was membrane-associated, only soluble protein was haemolytically active. The haemolytic activity was shown to be highly protease sensitive, heat labile, independent of divalent cations, and extremely dependent on protein concentration, consistent with a requirement for oligomerization as the mechanism of action. BlyA was highly purified from E. coli in a single step utilizing Triton X-114 phase partitioning. Genetic analysis of blyA and blyB mutants indicated that the stability, membrane association, and activity of BlyA was dependent on subtle changes in its sequence and on the BlyB protein. The bly genes were found to be expressed at a very low level in cultured B. burgdorferi .     

Cellular localization and export of the soluble haemolysin of Vibrio cholerae El Tor

Summary The cellular location of the haemolysin of Vibrio cholerae El Tor strain 017 has been analyzed. This protein is found both in the periplasmic space and the extracellular medium in Vibrio cholerae. However, when the cloned gene, present on plasmid pPM431, is introduced into E. coli K-12 this protein remains localized predominantly in the periplasmic space with no activity detected in the extracellular medium. Mutants of E. coli K-12 (tolA and tolB) which leak periplasmic proteins mimic excretion and release the haemolysin into the growth medium. Secretion of haemolysin into the periplasm is independent of perA (envZ) and in fact, mutants in perA (envZ) harbouring pPM431 show hyperproduction of periplasmic haemolysin. These results in conjunction with those for other V. cholerae extracellular proteins suggest that although E. coli K-12 can secrete these proteins into the periplasm, it lacks a specific excretion mechanism, present in V. cholerae, for the release of soluble proteins into the growth medium.     

Haemolysin genes of Vibrio cholerae: presence of homologous DNA in non-haemolytic O1 and haemolytic non-O1 strains

Abstract The structural gene for the haemolysin and two accessory genes from a Vibrio cholerae O1 El Tor strain have previously been cloned in Escherichia coli K-12 to give the plasmid pPM431. This plasmid has been used as a probe with a variety of O1 and non-O1 Vibrio cholerae strains to examine by Southern DNA hybridisations for the presence of homologous DNA. Such experiments show that the DNA homologous to that present in pPM431 is present in all of the 20 strains examined, whether they were haemolytic or non-haemolytic, implying that the genes were present but not expressed in non-haemolytic strains. Using a variety of restriction enzymes to cut the chromosomal DNA of different V. cholerae strains and probing with pPM431, it was possible to distinguish O1 and non-O1 strains, as well as haemolytic or non-haemolytic strains. This variability between hly⁺ and hly⁻ may be indicative of a change in the regulatory region of the haemolysin genes. The results also imply a high degree of homology of the haemolysin of O1 and non-O1 strains.     

Cloning and characterization of the genes encoding the haemolysin of Haemophilus ducreyi

We previously identified a heat- and protease-labile haemolytic activity expressed by Haemophilus ducreyi . In order to characterize the haemolysin at the molecular level, genomic DNA from H. ducreyi was probed with haemolysin genes from other Gram-negative organisms. The haemolysin genes of Proteus mirabilis hybridized to H. ducreyi DNA suggesting that the haemolysin of H. ducreyi is related to the Proteus/Serratia pore-forming family of haemolysins. Tn 916 mutagenesis was employed to isolate haemolysin-deficient mutants. Approximately 5000 Tn 916 transposon mutants were screened for the loss of haemolytic activity and two mutants were identified. One mutant, designated 35 000-1, was further characterized. Sequences flanking the Tn 916 element in strain 35 000-1 were employed to identify clones from a λDASHII library of H. ducreyi strain 35 000 DNA. A 13 kb insert from one lambda clone was selected for further study. This 13 kb fragment was able to both confer haemolytic activity to Escherichia coli and complement the haemolysin deficiency in strain 35 000-1. The haemolysin gene cluster was cloned from this 13 kb insert and two genes, designated hhdA and hhdB , were identified. The derived amino acid sequence of these genes demonstrated homology to the haemolysin and activation/secretion proteins of P. mirabilis and Serratia marcescens .     

The occurrence of duplicate lysyl-tRNA synthetase gene homologs in Escherichia coli and other procaryotes.

The lysyl-tRNA synthetase (LysRS) system of Escherichia coli K-12 consists of two genes, lysS, which is constitutive, and lysU, which is inducible. It is of importance to know how extensively the two-gene LysRS system is distributed in procaryotes, in particular, among members of the family Enterobacteriaceae. To this end, the enterics E. coli K-12 and B; E. coli reference collection (ECOR) isolates EC2, EC49, EC65, and EC68; Shigella flexneri; Salmonella typhimurium; Klebsiella pneumoniae; Enterobacter aerogenes; Serratia marcescens; and Proteus vulgaris and the nonenterics Pseudomonas aeruginosa and Bacillus megaterium were grown in AC broth to a pH of 5.5 or less or cultured in SABO medium at pH 5.0. These growth conditions are known to induce LysRS activity (LysU synthesis) in E. coli K-12. Significant induction of LysRS activity (twofold or better) was observed in the E. coli strains, the ECOR isolates, S. flexneri, K. pneumoniae, and E. aerogenes. To demonstrate an association between LysRS induction and two distinct LysRS genes, Southern blotting was performed with a probe representing an 871-bp fragment amplified from an internal portion of the coding region of the lysU gene. In initial experiments, chromosomal DNA from E. coli K-12 strain MC4100 (lysS+ lysU+) was double digested with either BamHI and HindIII or BamHI and SalI, producing hybridizable fragments of 12.4 and 4.2 kb and 6.6 and 5.2 kb, respectively. Subjecting the chromosomal DNA of E. coli K-12 strain GNB10181 (lysS+ delta lysU) to the same regimen established that the larger fragment from each digestion contained the lysU gene. The results of Southern blot analysis of the other bacterial strains revealed that two hybridizable fragments were obtained from all of the E. coli and ECOR collection strains examined and S. flexneri, K. pneumoniae, and E. aerogenes. Only one lysU homolog was found with S. typhimurium and S. marcescens, and none was obtained with P. vulgaris. A single hybridizable band was found with both P. aeruginose and B, megaterium. These results show that the dual-gene LysRS system is not confined to E. coli K-12 and indicate that it may have first appeared in the genus Enterobacter.     

Rapid and accurate identification of Escherichia coli K-12 strains.

A specific PCR for the identification of K-12 strains, based on the genetic structure of the O-antigen gene cluster (rfb) of Escherichia coli K-12, is described. The assay clearly differentiates E. coli K-12-derived strains from other E. coli strains used in the laboratory or isolated from human and animal clinical specimens, from food, or from environmental samples. Moreover, lineages of K-12 strains can be distinguished with a second PCR based on the same gene cluster. The method presents a useful tool in identifying K-12 for monitoring strains which are used as biologically safe vehicles in biotechnological research, development, and production processes.