Characterization of three group A klebicin plasmids: localization of their E colicin immunity genes

We have investigated the immunity to E colicins conferred by three group A klebicin plasmids. pP5a, which encodes klebicin A1-P5, like pClo-DF13, confers immunity to colicin E6 on Escherichia coli K12, whilst pP5b and pP3, which encode klebicins A2-P5 and A3-P3 respectively, both confer immunity to colicin E3. We have determined the restriction endonuclease and functional maps of the three group A klebicin plasmids. By sub-cloning and transposon mutagenesis we have investigated the relationship between the klebicin immunity and the E colicin immunity conferred by these plasmids. The colicin E6 and the klebicin A1 immunity are encoded by a single gene present on pP5a. The colicin E3 and the klebicin A2 immunity are encoded by a single gene present on pP5b. The colicin E3 and the klebicin A3 immunity are encoded by separate genes present on pP3. Recombinant pML8412, which is derived from the ColE6-CT14 plasmid and encodes colicin E6 immunity, confers klebicin A1-P5 immunity upon Klebsiella pneumoniae UNF5023. Recombinant pKC23, which is derived from the ColE3-CA38 plasmid and confers colicin E3 immunity, confers immunity to klebicin A2-P5, but not to klebicin A3-P3.     

Molecular characterization of the klebicin B plasmid of Klebsiella pneumoniae.

The nucleotide sequence of a bacteriocin-encoding plasmid isolated from Klebsiella pneumoniae (pKlebB-K17/80) has been determined. The encoded klebicin B protein is similar in sequence to the DNase pyocins and colicins, suggesting that klebicin B functions as a nonspecific endonuclease. The klebicin gene cluster, as well as the plasmid backbone, is a chimera, with regions similar to those of pore-former colicins, nuclease pyocins and colicins as well as noncolicinogenic plasmids. Similarities between pKlebB plasmid maintenance functions and those of the colicin E1 plasmid suggest that pKlebB is a member of the ColE1 plasmid replication family.     

Molecular Characterization of the Klebicin B Plasmid of Klebsiella pneumoniae

The nucleotide sequence of a bacteriocin-encoding plasmid isolated from Klebsiella pneumoniae (pKlebB-K17/80) has been determined. The encoded klebicin B protein is similar in sequence to the DNase pyocins and colicins, suggesting that klebicin B functions as a nonspecific endonuclease. The klebicin gene cluster, as well as the plasmid backbone, is a chimera, with regions similar to those of pore-former colicins, nuclease pyocins and colicins as well as noncolicinogenic plasmids. Similarities between pKlebB plasmid maintenance functions and those of the colicin E1 plasmid suggest that pKlebB is a member of the ColE1 plasmid replication family.     

Molecular characterization of pulA and its product, pullulanase, a secreted enzyme of Klebsielia pneumoniae UNF5023

The determined nucleotide sequence of the Klebsiella pneumoniae UNF5023 gene pulA comprises a single open reading frame coding for a 1090-residue precursor of the secreted protein pullulanase. The predicted sequence of this protein is highly homologous to that of pullulanase of Klebsiella aerogenes strain W70. However, the UNF5023 pullulanase lacks a collagen-like sequence present at the N-terminus of the mature W70 enzyme and differs further from the W70 pullulanase around residue 300 and at the C-terminus. Pullulanases with or without the collagen-like sequence could not be separated by gel electrophoresis under denaturing or non-denaturing conditions, and were unaffected by collagenase. A large central domain which is highly conserved in both UNF5023 and W70 polypeptides contains eight short sequences that are also found in amylases and iso-amylases. Linker mutations in the region of the UNF5023 pulA gene coding for this domain abolished catalytic activity without affecting transport of the polypeptide across the outer membrane. Hybrid proteins comprising at least the amino-terminal 656 residues of prepullulanase fused to alkaline phosphatase were partially localized to the cell surface, as judged by their accessibility to anti-pullulanase serum in immuno-fluorescence tests. On the basis of these results, we tentatively propose that secretion signals required for recognition and translocation across the outer membrane via the pullulanase-specific extension of the secretion pathway are located near the N-terminus of the pullulanase polypeptide.     

Cloning and characterization of the ColE7 plasmid

The 6.2 kb ColE7-K317 plasmid was mapped and the DNA fragments of the colicin E7 operon subcloned into pUC18 and pUC19. The size of the functional colicin E7 operon deduced by subcloning was 2.3 kb. The colicin E7 gene product was purified by carboxymethylcellulose chromatography. Both colicin E7 and E9 were demonstrated to exhibit a non-specific DNAase-type activity by in vitro biological assay. The molecular mass of colicin E7 was 61 kDa, as determined by SDS-PAGE. From DNA sequence data, the estimated sizes of the E7 immunity protein and the E7 lysis protein were 9926 Da and 4847 Da, respectively. Comparison of restriction maps and DNA sequence data suggests that ColE7 and ColE2 are more closely related than other E colicin plasmids.     

Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein

The previously uncharacterized third and fourth genes (pulE and pulF) of the pullulanase secretion gene operon of Klebsiella oxytoca strain UNF5023 are, respectively, predicted to encode a 55 kDa polypeptide with a putative nucleotide-binding site, and a highly hydrophobic 44 kDa polypeptide that probably spans the cytoplasmic membrane several times. Expression of pulE in minicells or under the control of a strong bacteriophage T7 promoter resulted in the production of a c. 58 kDa cytoplasmic protein. A representative PulE-beta-galactosidase hybrid protein created by Tnlac mutagenesis was also found mainly in the cytoplasm. These results are in line with the predicted absence from PulE of a region of sufficient hydrophobicity to function as a signal sequence. The PulF polypeptide could not be detected either in minicells or when the gene was transcribed from the T7 promoter, but the acquirement of three pulF-lacZ gene fusions that encoded hybrid proteins with relatively high levels of beta-galactosidase activity indicates that this gene can be transcribed and translated. Gene disruption experiments indicated that both pulE and pulF are required for pullulanase secretion in Escherichia coli K-12. Both proteins exhibit considerable homology throughout their entire lengths with other proteins involved in protein secretion, pilin assembly, conjugation and transformation competence in a variety of bacteria. In addition, PulE protein has consensus sequences found in a wide variety of nucleotide-binding proteins. This study completes the initial characterization of the pullulanase secretion gene operon, which comprises 13 genes that are all essential for the transport of pullulanase across the outer membrane.     

Distinct groups of plasmids correlated with bacteriocin production in Staphylococcus aureus

The genetic basis of bacteriocin (Bac) production by six strains of Staphylococcus aureus was examined. Gene transfer experiments (in which the plasmids were tagged with the erythromycin resistance transposon Tn551) and plasmid-elimination experiments by growth at 43 degrees C associated bacteriocin production with a particular plasmid in each strain. The Bac plasmids could be separated into two distinct groups: the first comprised plasmids larger than 40 kb, which did not specify immunity to bacteriocins; the second comprised small plasmids (8.0-10.4 kb) which also specified immunity to bacteriocins. The sequence relations among the small plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) were investigated by comparing restriction enzyme digest patterns and by hybridization. Plasmids pRJ10 and pRJ11 were indistinguishable and very closely related to plasmid pRJ9. Plasmid pRJ6, although different from the others, shared regions of sequence homology with them. No homology was found between plasmids pRJ6 or pRJ9 and the large Bac plasmids.     

Phage Associated Bacteriocins Reveal a Novel Mechanism for Bacteriocin Diversification in Klebsiella

Ninety-six isolates of Klebsiella pneumoniae and K. oxytoca were recovered from wild mammals in Australia. 14.6% of these bacteria produce killing phenotypes that suggest the production of bacteriocin toxins. Cloning and sequencing of the gene clusters encoding two of these killing phenotypes revealed two instances of a bacteriocin associated with a bacteriophage gene, the first such genetic organization described. The newly identified klebicin C gene cluster was discovered in both K. pneumoniae and K. oxytoca. The newly identified klebicin D gene cluster was detected in K. oxytoca. Protein sequence comparisons and phylogenetic inference suggest that klebicin C is most closely related to the rRNase group of colicins (such as colicin E4), while klebicin D is most closely related to the tRNase group of colicins (such as colicin D). The klebicin C and D gene clusters have similar genetic and regulatory organizations. In both cases, an operon structure is inferred consisting of a phage-associated open reading frame and klebicin activity and associated immunity genes. This novel bacteriophage/bacteriocin organization may provide a novel mechanism for the generation of bacteriocin diversity in Klebsiella.Reviewing Editor: Prof. David Guttman     

The purified colicin S8 is a multimeric protein.

Bacteriocins have been isolated both as simple proteins and as proteins in association with carbohydrates, lipids, etc. Colicins are commonly inducible and extracellular. Their molecular masses range from 30 to 90 kDa. Pure colicin S8 was obtained in three steps from supernatant of induced cells: (i) Ammonium sulfate precipitation; (ii) anion exchange chromatography; and (iii) phenyl-Sepharose hydrophobic chromatography, either by preparative or fast performance liquid chromatography (FPLC) analytical purification procedure. In our hands, purified colicin S8 was an aggregation of extremely related polypeptides. Composition of those active fractions was the same: five polypeptides of molecular weight around 55 kDa. Behavior on molecular filtration indicated a molecular weight higher than 200 kDa. Similar results were obtained when purification was carried out through FPLC. Producing strains contain a single plasmid that encodes colicin S8; in minicells, this plasmid was shown to specify a 60 kDa polypeptide. We conclude that more than one form of colicin S8 exists. The forms are structurally related and can be recognized by antibodies raised against one of the polypeptides. Consistent with this conclusion, comparison of peptides produced after hydrolysis with chlorosuccinamide indicated that the active proteins contained both shared and unique components.     

Periplasmic location of the pesticin immunity protein suggests inactivation of pesticin in the periplasm.

The pesticin activity and immunity genes on plasmid pPCP1 of Yersinia pestis were sequenced. They encoded proteins of 40 kDa (pesticin) and 16 kDa (immunity protein); the latter was found in the periplasm. The location of the immunity protein suggests that imported pesticin is inactivated in the periplasm before it hydrolyzes murein. Pesticin contains a TonB box close to the N-terminal end that is identical to the TonB box of colicin B. The DNA sequences flanking the pesticin determinant were highly homologous to those flanking the colicin 10 determinant. It is proposed that through these highly homologous DNA sequences, genes encoding bacteriocins may be exchanged between plasmids by recombination. In the case of pesticin, recombination may have destroyed the lysis gene, of which only a rudimentary fragment exists on pPCP1.     

Incompatibility and molecular relationships between small bacteriocinogenic plasmids of Staphylococcus aureus

The sequence relations between small bacteriocinogenic plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) of Staphylococcus aureus were investigated by comparing restriction maps and by hybridization. Plasmids pRJ10 and pRJ11 showed identical restriction maps, similar to that of pRJ9. The restriction map of pRJ6 differed from those of pRJ9 and pRJ10/pRJ11. Both groups of plasmids were shown to share a region of homology of at least 2.6 kb. The incompatibility relationships between them were also investigated by using plasmid derivatives tagged with transposon Tn551. Plasmids pRJ6 and pRJ9 proved to belong to different incompatibility groups.     

Cloning and expression in Escherichia coli of mercuric ion resistance coding genes from Zymomonas mobilis.

From a genomic library of Zymomonas mobilis prepared in Escherichia coli, two clones (carrying pZH4 and pZH5) resistant to the mercuric ion were isolated. On partial restriction analysis these two clones appeared to have the same 2.9 kb insert. Mercuric reductase activity was assayed from the Escherichia coli clone carrying pZH5 and it was Hg(2+)-inducible, NADH dependent and also required 2-mercaptoethanol for its activity. The plasmid pZH5 encoded three polypeptides, mercuric reductase (merA; 65 kDa), a transport protein (merT 18-17 kDa) and merC (15 kDa) as analysed by SDS-PAGE. Southern blot analysis showed the positive signal for the total DNA prepared from Hgr Z. mobilis but not with the Hgs strain which was cured for a plasmid (30 kb). These results were also confirmed by isolating this plasmid from Hgr Z. mobilis and transforming into E. coli. Moreover the plasmid pZH5 also hybridized with the mer probes derived from Tn21.     

Colicin U, a novel colicin produced by Shigella boydii.

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.     

Mutants of ColE1 plasmid with altered expression of the colicin E1 immunity gene

Mutagenesis with Tn1 transposon was used to isolate mutants of ColE1 plasmid with inactivated gene responsible for immunity to colicin E1. Cells containing such mutants synthesized active colicin E1 and were sensitive to its action. Spontaneous and UV-induced colicin synthesis was strongly changed in the mutants, as compared to the control. Mutations occurring outside the immunity gene, including those within the structural gene for colicin E1, could also affect the immunity gene expression.     

Construction of a new Streptococcus pneumoniae-Escherichia coli shuttle vector based on the replicon of an indigenous pneumococcal cryptic plasmid.

The nucleotide sequence of a cryptic plasmid (pRMG1) isolated from a type 1 Streptococcus pneumoniae has been determined and two recombinant plasmids, pRMGE1 and pRMGE2, bearing the pRMG1 replicon have been constructed. pRMGE2 is a shuttle vector for Escherichia coli and S. pneumoniae. The important characteristics of this cloning vector are: a size of 5.5 kb including a 1.4 kb fragment of pRMG1 (containing a double-stranded replication origin and an open reading frame encoding a putative replication initiation protein), a multicloning site, two antibiotic resistance markers for selection of plasmid containing cells, and blue-white colony screening in E. coli for identification of insert-containing plasmids.     

Characterisation of the ColE8 plasmid, a new member of the group E colicin plasmids

We have determined the restriction map of the ColE8-J plasmid after cloning it into the pBR322 vector. By subcloning and transposon mutagenesis we have localized the colicin immunity gene, the colicin structural gene, and lys, the region that determines MC sensitivity. In contrast to the ColE3-CA38 plasmid, the genes coding for colicin E8 production and immunity cannot be cloned on a single EcoRI fragment. Insertion of Tn5 transposons into the colicin structural gene region of the recombinant plasmid inactivated colicin production and MC sensitivity. Insertion of transposons into the lys region reduced colicin E8 production and MC induced lysis, the extent of which was dependent upon the precise site of insertion. We propose that the colicin E8 structural gene and lys must be transcribed from a common promoter situated proximal to the structural gene, whilst the colicin E8 immunity gene is transcribed from a second promoter. The lys region is responsible both for cell lysis after MC induction and positive regulation of colicin E8 synthesis.