Variant forms of matrix protein in Escherichia coli B/r bearing N plasmids

Plasmids of the N incompatibility group have been found to decrease or virtually eliminate the synthesis of the 36 500 dalton outer membrane matrix protein of their Escherichia coli B/r hosts (Iyer, R. (1977) Biochim. Biophys. Acta 470, 258–272 and Iyer, R., Darby, V. and Holland, I.B. (1978) FEBS Lett. 85, 127–132) or modify its composition. Although the 34 000 dalton tol G protein is slightly increased in some strains, it is identical in composition to the homologous protein from the plasmidless host. In three of five N⁺ strains, the synthesis of the modified matrix proteins depends on the temperature of cultivation of the strains in which they occur. The alterations to the matrix proteins are non-identical and do not affect the expression of several plasmid-coded functions including those of sensitivity to the N plasmid-specific filamentous bacteriophage IKe (Khatoon, H. and Iyer, R. (1971) Can. J. Microbiol. 17, 669–675), or their interbacterial transfer via conjugation to appropriate recipient strains. Thus, although the significance of the variant matrix proteins in N⁺ strains with respect to plasmid-mediated functions remains unclear, N plasmids nevertheless provide a convenient system which might be used to elucidate the events that precede the insertion of this protein into the outer membrane of E. coli B/r hosts.     

Variant forms of matrix protein in Escherichia coli B/r bearing N plasmids.

Plasmids of the N incompatibility group have been found to decrease or virtually eliminate the synthesis of the 36,500 dalton outer membrane matrix protein of their Escherichia coli B/r hosts (Iyer, R. (1977) Biochim. Biophys. Acta 470, 258--272 and Iyer, R., Darby, V. and Holland, I.B. (1978) FEBS Lett. 85, 127--132) or modify its composition. Although the 34,000 dalton tol G protein is slightly increased in some strains, it is identical in composition to the homologous protein from the plasmidless host. In three of five N+ strains the synthesis of the modified matrix proteins depends on the temperature of cultivation of the strains in which they occur. The alterations to the matrix proteins are non-identical and do not affect the expression of several plasmid-coded functions including those of sensitivity to the N plasmid-specific filamentous bacteriophage IKe (Khatoon, H. and Iyer, R. (1971) Can. J. Microbiol. 17, 669--675), or their interbacterial transfer via conjugation to appropriate recipient strains. Thus, although the significance of the variant matrix proteins in N+ strains with respect to plasmid-mediated functions remains unclear, N plasmids nevertheless provide a convenient system which might be used to elucidate the events that precede the insertion of this protein into the outer membrane of E. coli B/r hosts.     

Plasmids of incompatibility group P code for the capacity to propagate bacteriophage IKe.

Seven of eight plasmids of incompatibility group P were found to code for the capacity to propagate bacteriophage IKe in Escherichia coli. Six of the seven plasmids allowed propagation of IKe by one bacterial host (RG172) but not by another (RG176); the other plasmid allowed IKe propagation by both hosts. IKe propagation by a number of E. coli K-12 strains was quite variable. IKeh, an extended host range mutant of IKe, was found to plaque specifically on N+ and P+ strains.     

Isolation and characterisation of deletion mutants involving the transfer genes of P-group plasmids in Pseudomonas aeruginosa

Summary The P-group plasmids RP1 and R26 are recovered at low frequency following conjugal transfer to B3-lysogens of P. aeruginosa PAO. The rare carbenicillin-resistant transcipients that do arise are usually transfer-defective (Tra^-) and may show the loss of other plasmid borne functions, namely kanamycin-resistance (Km^r) and reduced plating of phage GlOl (Spp⁺). The four phenotypic classes that occur among the Tra^- derivatives are respectively, Tra^- (69–81%), Tra^- Spp^- (12–30%), Tra^- Km^s and Tra^- Km^s Spp^- (0.2–1%), of which the latter three are due to plasmid deletions. This is seen from the sizes of the plasmids carried by these bacteria and from the transductional analysis of the R26-derivatives. Thus, although R26 (MW=52×10⁶ daltons) is too large to be transduced by phage F116L (MW=40×10⁶), this is possible for its Tra^- Km^s and Tra^- Km^s Spp^- derivatives. The phenotypes and frequencies of the various transcipient classes suggests that the gene order Km..Tra..Spp occurs in both RP1 and R26, and that Spp is more closely linked to Tra than is Km. These conclusions are supported by the sizes of the plasmid mutants since deletions spanning the loci Km Tra Spp, Km Tra, and Tra Spp involve the loss of DNA of MW 8-17×10⁶, 5-13×10⁶ and 1-9×10⁶ daltons respectively.Whilst all the transcipients displayed the incompatibility properties of the parent plasmids (Inc⁺), only some retained plasmid surface exclusion (Sfx⁺). Moreover, a strict correlation existed between the Sfx and Spp phenotypes such that the transcipients were either wild type, Sfx^- Spp^-, or displayed an intermediate phenotype for both characters. This suggests that these phenotypes are controlled by closely linked genes or are different manifestations of the same gene function. The deletion map of these various markers in both RP1 and R26 therefore seems to be Km..Tra..Sfx/Spp..Inc.     

The membrane binding C-terminus of protein A fromStaphylococcus aureus affects its cellular localization and causes structural deformation when expressed inEscherichia coli

Protein A fromStaphylococcus aureus is a powerful diagnostic reagent and has several uses in human disease therapy. Expression in non-pathogenicEscherichia coli containing recombinant plasmids coding for this protein has increased its availability, but can reduce the stability of the plasmid-bearing host. By employing immune electron microscopy, we have determined thatE. coli containing stable plasmids coding for a truncated version of protein A, without the membrane binding site, secrete this protein through the cytoplasmic membrane and into the periplasmic space, where it accumulates.E. coli containing unstable plasmids, however, which code for the complete protein including the membrane-binding site, target the protein into the cytoplasmic membrane. This accumulation of protein A in theE. coli cytoplasmic membrane inhibits the formation of septa between dividing cells and results in aberrant elongated, multi-chromosomal forms.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Apparent variability in expression of the Bla+ phenotype of plasmid RP1 inPseudomonas acidovorans (RP1) strains

Expression of the Bla⁺ phenotype of the incompatibility group P-1 plasmid RP1 appears to be a variable phenomenon inPseudomonas acidovorans strains, although all plasmid-bearing strains examined synthesize the RP1-encoded TEM 2 β-lactamase. There is also evidence suggesting that plasmid-encoded alterations to the outer membrane could be affecting the degree of resistance observed to β-lactam drugs in at least some strains.     

Electron microscopic visualization of the arrangement of the two protein components of (Na + K)-ATPase

The information obtained by electron microscopic examination of highly purified membrane preparations of (Na⁺ + K⁺)-ATPase after freeze-fracturing or negative staining suggests the following conclusions. The catalytic 100 000 dalton protein component penetrates with its greater ‘globular’ mass the plasma membrane and protudes with its smaller mass from the protoplasmic surface by a stalked knob carrying the catalytic centre. The 40 000 dalton glycoprotein component is anchored in the membrane interior by a non-polar ‘fibrous’ side chain, whereas its major polar mass projects from the outer membrane surface forming a surface coat of ill-definable substructure.     

Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain

Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross-reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components.     

Construction of isogenic gonococcal strains varying in the presence of a 4.2-kilobase cryptic plasmid.

A 4.2-kilobase (kb) cryptic plasmid is present in 96% of isolates of Neisseria gonorrhoeae. An inability to construct isogenic derivatives which vary in the presence of the 4.2-kb plasmid has prevented the study of its function. We report a method to deliver an intact 4.2-kb plasmid into plasmidless gonococcal strains. The method involved transformation with novel 15.7-kb hybrid penicillinase-producing (Pcr) plasmids, which were cointegrates containing two copies of the 4.2-kb plasmid arranged in tandem direct repeat plus one copy of the 7.2-kb Pcr plasmid pFA3. When the 15.7-kb hybrid Pcr plasmids were introduced into a gonococcal recipient lacking evident plasmids, they dissociated at a relatively high frequency into plasmids identical to their parents: the 4.2-kb cryptic plasmid and pFA10 (a stable 11.5-kb plasmid containing one copy of each of the 7.2-kb Pcr plasmid pFA3 and the 4.2-kb cryptic plasmid pFA1). Curing strains of their Pcr plasmids resulted in isogenic strains which varied only in the presence of the 4.2-kb plasmid. The presence of the autonomously replicating 4.2-kb plasmid did not affect a number of tested phenotypes, including auxotype, antibiotic sensitivity, and frequencies of variation of outer membrane protein II. The interpretation of the functional significance of the 4.2-kb plasmid was complicated, however, by the additional finding that each of three tested plasmid-free strains contained a chromosomal fragment of about 1.6 kb that hybridized under moderate stringency with a 1.65-kb HinfI fragment of the 4.2-kb plasmid.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg[γ-³²P]ATP significant amounts of ³²P were incorporated into all these proteins. Incorporation of ³²P into the 15 000 dalton protein was moderately and ³²P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA²⁺ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca²⁺.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [³²P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Regulation of cell-wall morphology and growth encoded by plasmids in Shigella dysenteriae type 1

Shigella dysenteriae type 1, isolated locally, was found to contain six plasmids and these could be eliminated using SDS. A 70-kb plasmid was necessary to maintain the normal cell-wall morphology. Synthesis of nine major membrane proteins (90 to 40 kDa) was severely impaired in all-plasmid cured strains. Electron microscopy revealed a prominent separation between the outer and inner membranes of the cured strains, indicating that plasmid loss led to defects in the cell envelope. The growth rates of the strains having only the 70-kb plasmid and the plasmidless strain were 3- to 30-fold less than in the wild type strain.The authors are with the National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme XM, Beliaghata, Calcutta-700 010, India     

Functional analysis of the adsorption protein of two filamentous phages with different host specificities

The gene 3 coding for one minor coat protein (adsorption protein) of phage IKe was cloned into an expression plasmid and overproduced. The presence of a promoter for this gene could be demonstrated as well as the incorporation of the IKe gene 3 protein (g3p) into the cytoplasmic membrane of host cells. When 110 carboxy-terminal amino acids were deleted, the truncated protein was translocated across the cytoplasmic membrane into the periplasm. Thus the deleted amino acids bear a membrane anchor domain. In contrast to the partly homologous g3p of the Ff phages, IKe g3p did not alter the membrane properties of its host. IKe g3p was not incorporated into Ff phage particles in amounts detectable by our assays although the presence of IKe g3p may affect the efficiency of Ff phage production. The existence of a structural feature necessary for the specific recognition of the respective g3p during phage assembly is deduced.     

Bacteriophage X-2: a filamentous phage lysing IncX-plasmid-harbouring bacterial strains

Phage X-2, a filamentous rod about 950 nm in length, was isolated from sewage as plating on strains of Escherichia coli, Salmonella typhimurium or Serratia marcescens carrying either the IncX plasmid R6K, or the unique plasmid R775. Phage X-2 differs morphologically from a previously described very broad host range filamentous phage X which also lyses plasmid R6K-carrying strains and the phages differ in their resistance to inactivation by diethyl ether. Phage X-2 is serologically unrelated to phage X and the X-like phages IKe and I2-2. The adsorption site of the phage on the plasmid-bearing strains could not be determined but evidence implicating conjugative pili is presented.     

Conjugal Transfer in Lactic Streptococci of Plasmid-Encoded Insensitivity to Prolate- and Small Isometric-Headed Bacteriophages

Eight of 40 strains of Streptococcus lactis and S. lactis subsp. diacetylactis were able to conjugally transfer a degree of phage insensitivity to Streptococcus lactis LM0230. Transconjugants from one donor strain, S. lactis subsp. diacetylactis 4942, contained a 106-kilobase (kb) cointegrate plasmid, pAJ1106. The plasmid was conjugative (Tra⁺) and conferred phage insensitivity (Hsp) and lactose-fermenting ability (Lac) in S. lactis and Streptococcus cremoris transconjugants. The phage resistance mechanism was effective against prolate- and small isometric-headed phages at 30°C. In S. lactis transconjugants, the phage resistance mechanism was considerably weakened at elevated temperatures. A series of deletion plasmids was isolated from transconjugants in S. cremoris 4854. Deletion plasmids were pAJ2074 (74 kb), Lac⁺, Hsp⁺, Tra⁺; pAJ3060 (60 kb), Lac⁺, Hsp⁺; and pAJ4013 (13 kb), Lac⁺. These plasmids should facilitate mapping Hsp and tra genes, with the aim of constructing phage-insensitive strains useful to the dairy industry.     

Killing of Klebsiella pneumoniae mediated by conjugation with bacteria carrying antibiotic-resistance plasmids of the group N

Escherichia coli K-12 strains carrying any one of a number of different plasmids of the incompatibility group N have been found to kill Klebsiella pneumoniae, M5a1. A simple spot test is described that could be of value in diagnosing the presence of these plasmids. Killing is closely associated with the ability of the N⁺ strains to mate with M5a1 and all conjugation-deficient (Tra^?) mutants were also unable to kill M5a1 (Kil^?1). At a low frequency, some Kil^? mutants could be isolated that showed little or no transfer deficiency. Plasmids of the groups P and W which have been suspected (on other grounds) to specify conjugative systems interrelated in some manner to that specified by N plasmids, also kill M5a1 but to a lesser degree.     

Competition of plasmid-bearing Pseudomonas putida strains catabolizing naphthalene via various pathways in chemostat culture

Plasmid-carrying Pseudomonas putida strains degrade naphthalene through different biochemical pathways. The influence of various combinations of host bacteria and plasmids on growth characteristics and competitiveness of P. putida strains was studied in chemostat culture at a low dilution rate (D=0.05 h⁻¹) with naphthalene as the sole source of carbon and energy. Under naphthalene limitation, the plasmid-bearing strains degrading naphthalene that use catechol 1,2-dioxygenase for catechol oxidation (ortho pathway), were the most competitive. The strains bearing plasmids that control naphthalene catabolism via catechol 2,3-dioxygenase (meta pathway), were less competitive. Under these conditions the strain carrying plasmid pBS4, which encodes for naphthalene catabolism via gentisic acid, was the least competitive. Received: 24 February 1997 / Received revision: 22 May 1997 / Accepted: 25 May 1997     

R factor-mediated tetracycline resistance in Escherichia coli K12 dominance of some tetracycline sensitive mutants and relief of dominance by deletion

Summary Strains of Escherichia coli K12 heterozygous for the R100-1 tetracycline resistance region were constructed. They carried the wild-type Tet^r genes in the chromosome and single site Tet^s mutations on plasmids. Some heterozygotes could not express tetracycline resistance fully after induction. The mutant tet allele was thus partially dominant.When heterozygotes carrying the dominant tet mutant were plated on agar containing 50 g/ml tetracycline, mutants which grew normally occurred at a frequency of 1–4×10^-4. Analysis of these dominance relief mutants showed that in 53/56 isolates the dominant tet allele was lost forming either Tra⁺ or Tra^- deletion mutants of the plasmid. The mutation frequency was not affected either by the host chromosomal recA mutation or by the temperature of growth of the culture.     

Cloned structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12

Summary pTU 100 is a hybrid plasmid constructed by cloning a 7.5 Kb EcoRI fragment (carrying the wildtype ompA gene) onto pSC 101 (Henning et al., 1979). This plasmid confers sensitivity to phages Tull^* and K3h1 when present in an ompA host strain, due to the expression of the phage receptor protein II^* from the plasmid ompA ⁺ gene. Plasmid mutants have been isolated that have become resistant to one or both of these phages. Restriction endonuclease analysis and DNA-sequencing studies in these plasmids demonstrate that a BamHI site and two PvuII sites are located within the ompA gene. BamHI cuts the gene at a site corresponding to residue 227 within a total of 325 amino acid residues.Neither the wildtype ompA gene nor the BamHI fragment encoding the NH₂-terminal part of the protein (residues 1–227) could be transferred to a high copy number plasmid, presumably due to lethal overproduction of the protein or its NH₂-terminal fragment. However, the NH₂-terminal fragment derived from one of the ompA mutants of pTU100 could be transferred to the high copy number plasmid pBR322, and was expressed in the presence of the amber suppressors supD or supF. Under these conditions two new envelope proteins with apparent molecular weights of 30,000 and 24,000 were synthesized, and the cells became sensitive to phage TuII^*, indicating the presence of phage receptor activity in the outer membrane. The major, 24,000 dalton protein has the molecular weight expected of a protein comprising residues 1–227 of protein II^*. DNA-sequencing studies demonstrated that no termination codons are present in the DNA region immediately downstream from the BamHI site at residue 227 in this hybrid plasmid, and it is therefore likely that the 24,000-dalton protein arises from the posttranslational proteolytic cleavage of a larger polypeptide. The 30,000-dalton protein is a likely candidate for such a larger polypeptide. These results also demonstrate that the 98 CO₂H-terminal residues of wildtype protein II^* (resisdues 228–325) are not required either for the activity of the protein as a phage receptor or for its incorporation into the outer membrane.