Correlation Between the Expression of an Escherichia coli Cell Surface Protein and the Ability of the Protein to Bind to Lipopolysaccharide

The ompA gene of Escherichia coli codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (E. coli K-12 and two clinical isolates of E. coli) by transduction, the gene was expressed very poorly. Recombinants carrying “foreign” genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an E. coli K-12 recombinant carrying an unexpressed foreign ompA allele was subjected to two cycles of selection for an OmpA⁺ phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign ompA gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the ompA gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.     

Functions related to the receptor protein specified by the tsx gene of Escherichia coli

The receptor protein for the phage T6 and colicin K, coded by the tsx gene, facilitated the diffusion of all nucleosides and deoxynucleosides except cytidine and deoxcytidine through the outer membrane of Escherichia coli K-12 and Escherichia coli B. The tsx protein was coregulated with the nucleoside uptake system. Constitutive cytR and deoR mutants contained higher amounts of this protein than wild type strains. There was a good correlation between the initial rate of nucleoside uptake and the adsorption rate of phage T6. From the observation that nucleosides did not compete with each other in the translocation across the outer membrane and that they did not inhibit T6 adsorption it was concluded that the tsx protein forms a pore to which nucleosides have only little if any binding affinity.A major outer membrane protein specified by the ompA gene influenced the function of the tsx protein. Outer membranes of ompA mutants showed an enhanced binding of colicin K but the strains were colicin K insensitive (tolerant). The T6 phage adsorbed at the same rate and plated with the same efficiency as to ompA ⁺ strains. The uptake rate of thymidine and of adenosine was reduced by 16–33% in ompA mutants.The adsorption rate of phage T6 on mutants with altered lipopolysaccharide was the same or even higher than on wild type strains. However the plating efficiency was reduced ranging from 0–46%. Lipopolysaccharide plays no role in the primary adsorption of phage T6 but it is apparently required in a later step of the infection process.Non Standard Abbreviations LPS lipopolysaccharide - cAMP-CRP complex of cyclic adenosine 3,5-monophosphate (cAMP) and its receptor protein (CRP)     

The nature of ompA mutants of Escherichia coli K12 exhibiting temperature-sensitive bacteriophage resistance

Summary A class of ompA mutants of Escherichia coli, exhibiting temperature-sensitive resistance towards phages using the OmpA protein as receptor, was analysed. The mutants produce detectable levels of the protein at 42°C but not at 30°C (Manning and Reeves 1976). They were found to have a deletion (one isolate) or insertions (three isolates) upstream of the coding part of the ompA gene. Several previously characterized mutants possessing insertions or a deletion in the non-translated 5 area of the gene also exhibited a similar temperature-sensitive phage resistance. This cold-sensitive phenotype is explained in terms of the recent discovery that the stability of ompA mRNA is regulated by the rate of cell growth (Nilsson et al. 1984).     

meoA is the structural gene for outer membrane protein c of Escherichia coli K12

Summary The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Mel resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptorcomplex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c^*. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.     

Conjugation-deficient mutants of Escherichia coli distinguish classes of functions of the outer membrane OmpA protein

Summary Sixty-two E. coli mutants, selected as being deficient as recipients in F factor conjugation, are altered either in the amount or function of the outer membrane OmpA protein or in lipopolysaccharide structure. These two components may function together in conjugation, since the residual conjugation activity of a mutant lacking OmpA protein was unaffected by the additional presence of a lipopolysaccharide defect. Sixty of the strains carried mutations mapping to ompA, and these could be divided into classes depending on the amount of OmpA protein in their membranes. Representatives of these classes of mutant alleles failed to complement in diploids, indicating that they all affect the ompA structural gene and nearby sequences needed for its expression. The properties of these classes distinguish three groups of OmpA protein functions: 1) the structural function in the outer membrane in providing resistance to chelating agents and the hydrophobic antibiotic novobiocin, 2) the receptor functions in phage Tull^* and K3 infection, and 3) the functions of binding cells together during conjugation, facilitating the uptake of receptorbound colicin K or L, and allowing phage Ox2 to infect. Different cellular amounts or sites in OmpA protein are thus required for these three groups of functions.     

A cell wall-associated polysaccharide is required for bacteriophage adsorption to the Streptococcus thermophilus cell surface

Streptococcus thermophilus strain ST64987 was exposed to a member of a recently discovered group of S. thermophilus phages (the 987 phage group), generating phage-insensitive mutants, which were then characterized phenotypically and genomically. Decreased phage adsorption was observed in selected bacteriophage-insensitive mutants, and was partnered with a sedimenting phenotype and increased cell chain length or aggregation. Whole genome sequencing of several bacteriophage-insensitive mutants identified mutations located in a gene cluster presumed to be responsible for cell wall polysaccharide production in this strain. Analysis of cell surface-associated glycans by methylation and NMR spectroscopy revealed a complex branched rhamno-polysaccharide in both ST64987 and phage-insensitive mutant BIM3. In addition, a second cell wall-associated polysaccharide of ST64987, composed of hexasaccharide branched repeating units containing galactose and glucose, was absent in the cell wall of mutant BIM3. Genetic complementation of three phage-resistant mutants was shown to restore the carbohydrate and phage resistance profiles of the wild-type strain, establishing the role of this gene cluster in cell wall polysaccharide production and phage adsorption and, thus, infection.     

Outer Membrane Protein A (OmpA): A New Player in Shigella flexneri Protrusion Formation and Inter-Cellular Spreading

Outer membrane protein A (OmpA) is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92) and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the findings presented in this study.     

Isolation and Properties of Escherichia coli Mutants Which Are Nonpermissive for the Growth of Phage Lambda

By selecting survivors of λ phage infection, mutants of Escherichia coli K12 that block reproduction cycle of the phage have been isolated. Fourteen of these phage-tolerant mutants (lam mutants) were chosen and characterized biochemically and genetically. It was shown that these mutants were tolerant to infection by all the lambdoid phages, except for few cases, but they were susceptible to infection by a non-lambdoid temperate phage (φ299), P1 or T phages. The mutants can be divided into at least three groups: (1) A mutant (lam 16) strain that seems to block normal penetration of phage DNA: (2) Three mutant (lam 64, lam 67 and lam 71) strains that block an “early” step(s) of phage growth, including phage DNA synthesis: (3) Six mutant (lam 24, lam 25, lam 26, lam 27, lam 646 and lam 6) strains that block normal functioning of the gene E products and produce unusual head structures. Some lambdoid phages and λ mutants that overcome the interference by the lam mutations have been obtained, and were used as tools for characterizing the host mutations. Two (lam 12 and lam 13) mutant strains and one (lam 1) mutant were inferred as affecting the expression of “late” genes, and early gene, respectively, by this test.     

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA^- strain were G:C → T:A transversions, occurring within the sequence which in recA⁺ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.     

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.     

Functional Interaction of the tonA/tonB Receptor System in Escherichia coli

Host range mutants of phage T1 (T1h), which productively infected tonB mutants of Escherichia coli, were isolated. The phage mutants were inactivated by isolated outer membranes of E. coli in contrast to the wild-type phage, which only adsorbed reversibly. For the infection process, the tonB function is apparently only required for the irreversible adsorption of the phage T1, but not for the transfer of the phage DNA through the outer membrane and the cytoplasmic membrane of the cell. Mutants of the tonA gene expressing normal amounts of outer membrane receptor proteins were isolated and found to be partially sensitive to phage T5 and resistant to the phages T1 and T1h, colicin M, and albomycin and unable to take up iron as a ferrichrome complex. One tonA mutant remained partially sensitive to T5, colicin M, and albomycin and supported growth of T1h (not of T1) with the same plating efficiency as the parent strain. Only a small region of the tonA receptor protein seems to function for all the very different substrates. A newly isolated host range mutant of T5 (T5h) adsorbed faster to tonA(+) cells than did wild-type T5 and infected tonA missense mutants resistant to wild-type T5. The interplay of the tonA with the tonB function was observed with phage T5 infection, although T5 required only the tonA receptor. Ferrichrome inhibited plaque formation of T5 only when plated on tonB mutants. Adsorption of T5 to cells in liquid medium was influenced by ferrichrome as follows: complete inhibition by 0.1 muM ferrichrome with tonB mutants, not more than 35% inhibition by 1 to 100 muM ferrichrome with the tonB(+) parent strain in the presence of glucose as energy source, and 90% inhibition by 1 muM ferrichrome with partially starved parent cells. We conclude that there exist different functional states of the receptor protein that depend on the energy state of the cell and the tonB function. The latter seems to be required only for translocation processes with outer membrane proteins involved.     

A method for the isolation of phage mutants altered in their response to lysogenic induction

Summary Phage cl ⁺ gives clear plaques whereas phage cIind ^- gives turbid plaques on a lawn of a mutant strain of E. coli K12. This strain, called STS, carries mutation spr in a tif sfi genetic background. I hypothesize that upon temperate phage infection, STS bacteria spontaneously inactivate phage repressor by the same mechanism involved in normal lysogenic induction which results in obligatory lytic growth of ⁺. The use of the STS mutant facilitates the isolation and genetic analysis of phage mutants with an abnormal response to lysogenic induction.     

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.     

Phenotypes ofdnaA mutants ofEscherichia coli sensitive to phenothiazine derivatives

The activation of DnaA protein by cardiolipin is inhibited by fluphenazinein vitro. We therefore examined the sensitivity of temperature-sensitivednaA mutants ofEscherichia coli to fluphenazine and other phenothiazine derivatives. Among the eightdnaA mutants tested,dnaA5, dnaA46 dnaA602, anddnaA604, mutants with mutations in the putative ATP binding site of DnaA protein, showed higher sensitivities to phenothiazine derivatives than did the wild-type strain. ThednaA508 anddnaA167 mutants, which have mutations in the N-terminal region of DnaA protein, also showed higher sensitivities to phenothiazine derivatives. On the other hand, thednaA204 anddnaA205 mutants, with lesions in the C-terminal region of the DnaA protein, showed the same sensitivity to phenothiazine derivatives as the wild-type strain. Complementation analysis with a plasmid containing the wild-typednaA gene and phage P1-mediated transduction confirmed thatdnaA mutations are responsible for these sensitivity phenotypes.     

Isolation and characterization of bacteriophage FC3-10 from Klebsiella spp.

FC3-10 is a Klebsiella spp. specific bacteriophage isolated on a rough mutant (strain KT707, chemotype Rd) of K. pneumoniae C3. The bacteriophage receptor for this phage was shown to be the low-molecular mass lipopolysaccharide (LPS) fraction (LPS-core oligosaccharides), specifically the heptose content of the LPS inner-core. This is the first phage isolated on Klebsiella, the receptor for which is the LPS-core. This phage was unable to plate on Salmonella typhimurium LPS mutants with chemotypes Rd2 or Re showing incomplete or no heptose content on their LPS-core, respectively. Spontaneous phage-resistant mutants from different Klebsiella strains were deep-rough LPS mutants or encapsulated revertants from unencapsulated mutant strains.     

Temperature-sensitive,lipopolysaccharide-deficient mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium LT2, which were temperature-sensitive for lipopolysaccharide (LPS) synthesis, were isolated from a galE ^- strain based on their resistance to phage C21 and sensitivity to sodium deoxycholate at 42°C. They produced LPS of chemotype Rc at 30°C and deep-rough LPS at 42°C. P22-mediated transductional analysis showed that the mutations responsible for temperature sensitivity are located in the rfa cluster where several genes involved in the synthesis of the LPS core are mapped. A plasmid, carrying rfaC, D and F genes of Escherichia coli K-12, complemented these mutations. These genes are responsible for the synthesis of the inner-core region of the LPS molecule. This indicates that genetic defects in these temperature-sensitive mutants affect the inner-core region of LPS.     

Isolation and characterization of motile Escherichia coli mutants resistant to bacteriophage chi.

Four mutants of Escherichia coli that are resistant to the flagellotropic phage chi, but are motile, were isolated. When they were observed in liquid culture bylight microscopy, one mutant exhibited circular movement and another tumbled at high frequency on the surface of a glass slide. The remaining two mutants moved normally. None of these mutants adsorbed the wild-type strain of chi. P1 transduction revealed that the mutation sites of these four mutants were more than 97% contransducible with a site in hag, the structural gene for flagellin. When flagellins of these mutants were chromatographed on a diethylaminoethyl-cellulose column, two eluted slower and one eluted slightly faster than the flagellin of the parental strain. The other flagellin eluted at the same position as that of the parent. Host range mutants of phage chi, which could infect these bacterial mutants, were isolated.     

Isolation and Characterization of LPS Mutants of Actinobacillus pleuropneumoniae Serotype 1

The major adhesin of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, has been previously identified as lipopolysaccharide (LPS). The purpose of the present study was to isolate and characterize A. pleuropneumoniae LPS mutants. Screening of LPS mutants was performed with colony dot and sensitivity to novobiocin. One mutant obtained by colony dot (F19) and one mutant selected for its increased sensitivity to novobiocin (33.1) did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 O-antigen compared with the parent strain. Mutants F19 and 33.1 did not express high-molecular-mass LPS bands as determined in silver-stained SDS-PAGE gels. The core-lipid A region of mutant 33.1 and of the parent strain had similar relative mobilities and reacted with serum from a pig experimentally infected with the serotype 1 reference strain of A. pleuropneumoniae, while the same region in mutant F19 showed faster migration and did not react with this serum. Use of piglet tracheal frozen sections indicated that mutant F19 was able to adhere to piglet trachea as well as the parent strain, while mutant 33.1 adhered [half as much as] the parent strain. Finally, both LPS mutants were markedly less virulent in mice than the parent strain. Taken together, our observations support the idea that LPS is an important virulence factor of A. pleuropneumoniae. Received: 23 December 1996 / Accepted: 19 February 1997