Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli

Expression of cloned gene E of bacteriophage PhiX174 induces lysis by formation of a transmembrane tunnel structure in the cell envelope of Escherichia coli. Ultrastructural studies of the location of the lysis tunnel indicate that it is preferentially located at the septum or at polar regions of the cell. Furthermore, the diameter and shape of individual tunnel structures vary greatly indicating that its structure is not rigid. Apparently, the contours of individual lysis tunnels are determined by enlarged meshes in the peptidoglycan net and the force produced at its orifice, by the outflow of cytoplasmic content. Once the tunnel is formed the driving force for the lysis process is the osmotic pressure difference between cytoplasm and medium. During the lysis process areas of the cytoplasmic membrane which are not tightly attached to the envelope are extended inward by the negative pressure produced during lysis. After cell lysis external medium can diffuse through the lysis tunnel filling the inner cell space of the still rigid bacterial ghosts.     

Using fusions with luxAB from Vibrio harveyi MAV to quantify induction and catabolite repression of the xyl operon in Staphylococcus carnosus TM300

Abstract The luxA,B genes from the Gram-negative marine bacterium Vibrio harveyi MAV were used in Staphylococcus carnosus TM300 as a reporter system for regulated expression of xylose utilization. The luciferase genes were fused to the xyl operon from Staphylococcus xylosus C2a. Expression of bioluminescence was induced through addition of xylose and repressed in the presence of glucose. A method to quantitate bioluminescence directly from the culture is described.     

Isolation and characterization of a virulent bacteriophage from Staphylococcus carnosus

Abstract A virulent bacteriophage, øSK311, was isolated from Staphylococcus carnosus , an organism used as a starter culture for the production of dry sausage. Electron microscopic studies revealed that this bacteriophage showed some morphological similarities with the Escherichia coli phages T4 and λ. The host range of øSK311 extends over 9 different staphylococcal species. A phage-resistant mutant of S. carnosus could be isolated. Cells of this mutant exhibited a capsule-like structure. The DNA of øSK311 possesses a G + C content of 31.4 mol% and appears to be highly modified.     

Cloning and nucleotide sequence of the signal peptidase II (lsp)-gene from Staphylococcus carnosus

Abstract Staphylococcus carnosus TM300 is able to synthesize at least seven lipoproteins with molecular masses between 15 and 45 kDa; the proteins are located in the membrane fraction. It can be concluded that this strain also posesses the enzymes involved in lipoprotein modification and prolipoprotein signal peptidase (signal peptidase II) processing. The gene encoding the prolipoprotein signal peptidase, lsp , from Staphylococcus carnosus TM300 was cloned in Escherichia coli and sequenced. The deduced amino acid sequence of the Lsp showed amino acid similarities with the Lsp's of S. aureus , Enterobacter aerogenes, E. coli , and Pseudomonas fluorescens . The hydropathy profile reveals four hydrophobic segments which are homologous to the putative transmembrane regions of the E. coli signal peptidase II. E. coli strains carrying lsp of S. carnosus exhibited an increased globomycin resistance.     

Changes in host cell phospholipid composition of φX174 gene E product

Abstract Expression of bacteriophage φX174 gene E from plasmid pUH51 induced lysis of Escherichia coli . Before onset of bacterial lysis, cellular phospholipase activity was induced due to the presence of gene E product within the cells. By comparison of the lytic behaviour of phospholipase-negative E. coli strains with the corresponding wild-type strain it was found that neither the action of detergent-resistant phospholipase A nor of detergent-sensitive phospholipase were essential for the lysis-inducing properties of the gene E product. It was concluded that induction of phospholipases after expression of the φX174 gene E was a consequence of membrane perturbation caused by the integration of the gene E product into the cytoplasmic membrane of E. coli .     

Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism

AIMS: The study was conducted with an aim to optimize the transformation efficiency of the Gram-positive bacterium Staphylococcus carnosus to a level that would enable the creation of cell surface displayed combinatorial protein libraries. METHODS AND RESULTS: We have thoroughly investigated a number of different parameters for: (i) the preparation of electrocompetent cells; (ii) the treatment of cells before electroporation; (iii) the electroporation step itself; and (iv) improved recovery of transformed cells. Furthermore, a method for heat-induced inactivation of the host cell restriction system was devised to allow efficient transformation of the staphylococci with DNA prepared from other species, such as Escherichia coli. Previously described protocols for S. carnosus, giving transformation frequencies of approximately 10(2) transformants per transformation could be improved to reproducible procedures giving around 10(6) transformants for a single electroporation event, using plasmid DNA prepared from either S. carnosus or E. coli. The transformed staphylococcal cells were analysed using flow cytometry to verify that the entire cell population retained the introduced plasmid DNA and expressed the recombinant protein in a functional form on the cell surface at the same level as the positive control population. CONCLUSIONS: The results demonstrate that the transformation frequency for S. carnosus could be dramatically increased through optimization of the entire electroporation process, and that the restriction barrier for interspecies DNA transfer, could be inactivated by heat treatment of the cells prior to electroporation. SIGNIFICANCE AND IMPACT OF THE STUDY: The generation of large combinatorial protein libraries, displayed on the surface of S. carnosus can be envisioned in the near future, thus dramatically improving the selection compared with the traditional biopanning procedure used in phage display.     

Suppression of an Escherichia coli secAts mutant by a gene cloned from Staphylococcus carnosus

Escherichia coli mutant MM52 (secA(ts)) was transformed with a cosmid library from Staphylococcus carnosus, and a recombinant cosmid (pBO23) allowing growth at the non-permissive temperature (42 degrees C) was isolated. pBO23 also restored the growth defects of E. coli mutants IQ85 (secY(ts)) and IT41 (lep(ts)). Nucleotide sequencing revealed that the DNA fragment responsible for the suppression effect codes for a S. carnosus protein highly homologous to the ribosomal protein L13 of E. coli. The staphylococcal L13 protein was efficiently incorporated into E. coli ribosomes. Possible explanations for the effect of this polypeptide on the growth of temperature-sensitive E. coli secretion mutants are discussed.     

Two-stage model for integration of the lysis protein E of ΦX174 into the cell envelope of Escherichia coli

Abstract: As a tool for determining the topology of the small, 91-amino acid ΦX174 lysis protein E within the envelope complex of Escherichia coli , a lysis active fusion of protein E with streptavidin (E-FXa-StrpA) was used. The E-FXa-StrpA fusion protein was visualised using immune electron microscopy with gold-conjugated anti-streptavidin antibodies within the envelope complex in different orientations. At the distinct areas of lysis characteristic for protein E, the C-terminal end of the fusion protein was detected at the surface of the outer membrane, whereas at other areas the C-terminal portion of the protein was located at the cytoplasmic side of the inner membrane. These results suggest that a conformational change of protein E is necessary to induce the lysis process, an assumption supported by proteinase K protection studies. The immune electron microscopic data and the proteinase K accessibility studies of the E-FXa-StrA fusion protein were used for the working model of the E-mediated lysis divided into three phases: phase 1 is characterised by integration of protein E into the inner membrane without a cytoplasmic status in a conformation with its C-terminal part facing the cytoplasmic side; phase 2 is characterised by a conformational change of the protein transferring the C-terminus across the inner membrane; phase 3 is characterised by a fusion of the inner and outer membranes and is associated with a transfer of the C-terminal domain of protein E towards the surface of the outer membrane of E. coli.     

Cloning and expression of Staphylococcus epidermidis urease gene sequences in Staphylococcus carnosus

The urease gene sequences of Staphylococcus epidermidis CNS23 were cloned and expressed in Staphylococcus carnosus TM300. In vitro translation of the cloned sequences revealed four polypeptides (60, 17, 11 and 7.5 kDa) that were associated with enzyme activity. Southern hybridisation experiments showed high homologies with the urease genes of Staphylococcus saprophyticus.     

Characterization of the molybdate transport system ModABC of Staphylococcus carnosus

Transposon mutagenesis of Staphylococcus carnosus led to the identification of three genes, modABC, which encode an ABC transporter that is involved in molybdate transport. It was shown by [¹⁴C]palmitate labeling that ModA represents a lipoprotein that in gram-positive bacteria is the counterpart of the periplasmic binding proteins of gram-negative organisms. The sequence characteristics identify ModB as the integral-membrane, channel-forming protein and ModC as the ATP-binding energizer for the transport system. Mutants defective in modABC had only 0.4% of the wild-type nitrate reductase activity. Molybdate at a non-physiologically high concentration (100 μM) fully restored nitrate reductase activity, suggesting that at least one other system is able to transport molybdate, but with lower affinity. The expression of modA (and most likely of modBC) was independent of oxygen and nitrate. To date, there are no indications for molybdate-specific regulation of modABC expression since in a modB mutant, modA expression was unchanged in comparison to the wild-type. Received: 5 February 1999 / Accepted: 31 May 1999     

φX174 lysis requires slyD, a host gene which is related to the FKBP family of peptidyl-prolyl cis-trans isomerases

Abstract: Recessive mutations in the slyD (sensitivity to ly sis) gene were isolated by selecting for survival after induction of the cloned lysis gene E of bacteriophage φX174 [1]. The slyD ⁻ mutation, transduced into the normal φX174 host, Escherichia coli C, confers an absolute block on the plaque-forming ability of the wild-type phage, indicating that slyD is required for E function. slyD encodes a protein with 196 residues. A segment corresponding to the first 142 residues of the predicted SlyD protein has significant similarity throughout its length to the FKBP family of peptidyl-prolyl cis-trans isomerases, or rotamases. The C-terminal 46 codons of slyD encode a remarkable histidine-rich peptide which is a metal-binding domain [2]. This sequence is dispensable for slyD function in E -mediated lysis. Although there is no obvious phenotype associated with the slyD ⁻ genotype other than the resistance to E -mediated lysis, overexpression of slyD causes cells to filament and to increase significantly in diameter. Mutations in φX174 can restore the plaque-forming ability of the phage on a slyD ⁻ host. These pos ( p lates on s lyD) mutants plate on E. coli C wild-type and slyD ⁻. A model for SlyD involvement in E function and the role of SlyD in the cell is discussed.     

Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli

Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174. Gene E expression is tightly controlled by the rightward lambda pR promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12. The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines. Expression of gene E is stringently repressed at temperatures up to 30 degrees C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30 degrees C due to thermal inactivation of the cI857 repressor. As a consequence, the production of ghosts requires that bacteria have to be grown at 28 degrees C before the lysis process is induced. In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the lambda pR promoter/cI857 repressor system, allowing pathogens to grow at 37 degrees C before induction of lysis. In this study we describe a mutation in the lambda pR promoter, which allows stringent repression of gene E expression at temperatures up to 36 degrees C, but still permits induction of cell lysis at 42 degrees C.     

Use of a lytic enzyme system from Cytophaga sp. in the lysis of Gram-positive bacteria

A lytic enzyme system from Cytophaga sp. has been used for lysis of the Gram-positive bacteria, Bacillus and Corynebacterium. The optimum pH and temperature for the lytic reaction were 9.2 and 50°C, respectively. The effect of substrate and enzyme concentration have also been studied. Protein release was followed and the potential of using bacteriolytic enzymes for large-scale cell lysis and release of intracellular material is discussed.     

Improved method for electroporation of Staphylococcus aureus

We have developed a significantly improved method for the electroporation of plasmid DNA into Staphylococcus aureus. The highest transformation efficiency achieved with this procedure was 4.0 x 10(8) transformants per microgram of plasmid pSK265 DNA. This represents a 530-fold improvement over the previously reported optimum efficiency of 7.5 x 10(5) transformants per microgram of plasmid DNA after electroporation of S. aureus cells [9]. Identical results were obtained when electrocompetent cells, which had been stored frozen at -80 degrees C, were used. The improved efficiency is due primarily to the use of a modified medium (designated as B2 medium) and secondarily to the use of 0.1-cm cuvettes. Several other plasmids (pI258, pMH109, and pSK270) were also electrotransformed into competent cells using our procedure, and for each plasmid, the transformation efficiency was significantly reduced compared to that observed when pSK265 DNA was used. With respect to plasmid pI258, the transformation efficiency was 3500-fold higher than that reported previously for transformation of this plasmid into S. aureus RN4220 [9]. The optimized electroporation procedure was less successful in transforming other staphylococci. Electrocompetent cells of S. aureus ATCC 29213 and S. epidermidis ATCC 12228 produced 5.5 x 10(5) and 5 x 10(3) transformants per microgram of pSK265 DNA, respectively.