DNA sequence heterogeneity in the genes of T-even type Escherichia coli phages encoding the receptor recognizing protein of the long tail fibers

Summary Genes (g) 36 and 37 code for the proteins of the distal half of the long tail fibers of phage T4, gene product (gp) 35 links the distal half to the proximal half of this fiber. The receptor, lipopolysaccharide, most likely is recognized by gp37. Using as probe a restriction fragment consisting of most of g36 and g37 of phage T4 the genes corresponding to g35, g36, and g37 of phages T2 and K3 (using the E. coli outer membrane proteins OmpF and OmpA, respectively, as receptors) have been cloned into plasmid pUC8. Partial DNA sequences of g37 of phage K3 have been determined. One area, corresponding to residues 157 to 210 of the 1026 residue gp37 of phage T4, codes for an identical sequence in phage K3. Another area corresponds to residues 767 to 832 of the phage T4 sequence. Amino acid residues 767 to 832 of the phage T4 sequence are almost identical in both phage proteins while the remainder is rather different. DNAs of T2, T4, T6, another T-even type phage using protein Tsx as a receptor, and 10 different T-even type phages using the OmpA protein as a receptor have been hybridized with restriction fragments covering various parts of the g37 area of phage K3. With probably only one exception all of the 13 phages tested possess unique genes 37 and within the majority of these, sequences highly homologous to parts of g37 of K3 are present in a mosaic type fashion. Other regions of these genes 37 did not show any homology with the K3 probes; in case of the OmpA specific phage M1 absence of homology was evident in most of its g37 even including the area that should serve for recognition of the cellular receptor. In sharp contrast to this situation it was found that a major part of the gene (g23) coding for the major capsid protein is rather highly conserved in all phages studied. The extreme variability in sequences existing in genes 37 might be a consequence of phages during evolution being able to more or less drastically change their receptor specifities.     

Genetic Definition of Two Functional Elements in a Bacteriophage T4 Host-Range ``cassette''''

Gene 37 of T4 encodes the major subunit of the distal half of the tail fiber. The distal tip of the fiber, comprised of the carboxy-terminal ends of two molecules of gene 37 product (gp37), carries the principal determinant of the phage host range. The gp37 carboxyl termini recognize the bacterial surface during infection, and, in addition, include a site required for interaction with the product of gp38 during distal half-fiber assembly. In the absence of interaction with gp38, gp37 polypeptides do not dimerize. Eleven temperature-sensitive mutants with defects located near the promoter-distal end of gene 37 were tested at nonpermissive temperatures for production of an antigen that is diagnostic of distal half-fiber assembly. Six of the mutations prevent distal half-fiber assembly. The other five allow assembly of distal half fibers, which combine with proximal half fibers and attach to phage particles, but the resulting phage do not adsorb to bacteria. These two classes of mutations define two adjacent but separate genetic regions, corresponding to two different functional domains in gp37. These two regions and the neighboring gene 38 comprise a functional unit that can be considered as a host-range "cassette," with features that are strikingly similar to corresponding functional units in other unrelated as well as related phages.     

T-even-type bacteriophages use an adhesin for recognition of cellular receptors

Protein 38 of the Escherichia coli phage T4 is thought to be required catalytically for the assembly of the long tail fibers of this phage. It is shown that this protein of phage T2 and the T-even-type phage K3 and Ox2 act differently. It was found that NH2-terminal fragments of the protein, expressed from cloned fragments of gene 38 of phage K3, bind to gene 38 amber mutants of phage T2. Such phage or T2 gene 38 amber mutants, grown on a non-permissive host, possess a complete set of six tail fibers but are non-infectious. Both types of non-infectious phage could be repaired by incubation with an extract of cells harboring a cloned gene 38 of a host range mutant of phage K3, K3hx. The repaired phages had the host range of K3hx and not of T2. Immuno-electron microscopy showed that protein 38 is located at the free ends of the long tail fibers of phages T2, K3 and Ox2. The protein serves the recognition of the cellular receptor, i.e. it acts as an adhesin.     

Characterization of the distal tail fiber locus and determination of the receptor for phage AR1, which specifically infects Escherichia coli O157:H7

Phage AR1 is similar to phage T4 in several essential genes but differs in host range. AR1 infects various isolates of Escherichia coli O157:H7 but does not infect K-12 strains that are commonly infected by T4. We report here the determinants that confer this infection specificity. In T-even phages, gp37 and gp38 are components of the tail fiber that are critical for phage-host interaction. The counterparts in AR1 may be similarly important and, therefore, were characterized. The AR1 gp37 has a sequence that differs totally from those of T2 and T4, except for a short stretch at the N terminus. The gp38 sequence, however, has some conservation between AR1 and T2 but not between AR1 and T4. The sequences that are most closely related to the AR1 gp37 and gp38 are those of phage Ac3 in the T2 family. To identify the AR1-specific receptor, E. coli O157:H7 was mutated by Tn10 insertion and selected for an AR1-resistant phenotype. A mutant so obtained has an insertion occurring at ompC that encodes an outer membrane porin. To confirm the role of OmpC in the AR1 infection, homologous replacement was used to create an ompC disruption mutant (RM). When RM was complemented with OmpC originated from an O157:H7 strain, but not from K-12, its AR1 susceptibility was fully restored. Our results suggest that the host specificity of AR1 is mediated at least in part through the OmpC molecule.     

Single mutations in a gene for a tail fiber component of an Escherichia coli phage can cause an extension from a protein to a carbohydrate as a receptor

The T-even type Escherichia coli phage Ox2 recognizes the outer membrane protein OmpA as a receptor. This recognition is accomplished by the 266 residue protein 38, which is located at the free ends of the virion's long tail fibers. Host-range mutants had been isolated in three consecutive steps: Ox2----Ox2h5----Ox2h10----Ox2h12, with Ox2h12 recognizing the outer membrane protein OmpC efficiently and having lost some affinity for OmpA. Protein 38 consists, in comparison with these proteins of other phages, of two constant and one contiguous array of four hypervariable regions; the alterations leading to Ox2h12 were all found within the latter area. Starting with Ox2h12, further host-range mutants could be isolated on strains resistant to the respective phage: Ox2h12----h12h1----h12h1.1----h12h1.11----h12 h1.111. It was found that Ox2h12h1.1 (and a derivative of Ox2h10, h10h4) probably uses, instead of OmpA or OmpC, yet another outer membrane protein, designated OmpX. Ox2h12h1.11 was obtained on a strain lacking OmpA, -C and -X. This phage could not grow on a mutant of E. coli B, possessing a lipopolysaccharide (LPS) with a defective core oligosaccharide; Ox2h12h1.111 was obtained from this strain. It turned out that the latter two mutants used LPS as a receptor, most likely via its glucose residues. Selection for resistance to them in E. coli B (ompA+, ompC-, ompX-) yielded exclusively LPS mutants, and in another strain, possessing OmpA, C and X, the majority of resistant mutants were of this type. Isolated LPS inactivated the mutant phages very well and was inactive towards Ox2h12. By recombining the genes of mutant phages into the genome of parental phages it could be shown that the phenotypes were associated with gene 38. All mutant alterations (mostly single amino acid substitutions) were found within the hypervariable regions of protein 38. In particular, a substitution leading to Ox2h12h1.11 (Arg170----Ser) had occurred at the same site that led to Ox2h10 (His170----Arg), which binds to OmpC in addition to OmpA. It is concluded that not only can protein 38 gain the ability to switch from a protein to a carbohydrate as a receptor but can do so using the same domain of the polypeptide.     

Escherichia coli K-12 outer membrane protein (OmpA) as a bacteriophage receptor: analysis of mutant genes expressing altered proteins.

The outer membrane protein OmpA of Escherichia coli K-12 serves as a receptor for a number of T-even-like phages. We have isolated a series of ompA mutants which are resistant to such phages but which still produce the OmpA protein. None of the mutants was able to either irreversibly or reversibly bind the phage with which they had been selected. Also, the OmpA protein is required for the action of colicins K and L and for the stabilization of mating aggregates in conjugation. Conjugal proficiency was unaltered in all cases. Various degrees of colicin resistance was found; however, the resistance pattern did not correlate with the phage resistance pattern. DNA sequence analyses revealed that, in the mutants, the 325-residue OmpA protein had suffered the following alterations: Gly-65----Asp, Gly-65----Arg, Glu-68----Gly, Glu-68----Lys (two isolates), Gly-70----Asp (four isolates), Gly-70----Val, Ala-Asp-Thr-Lys-107----Ala-Lys (caused by a 6-base-pair deletion), Val-110----Asp, and Gly-154----Ser. These mutants exhibited a complex pattern of resistance-sensitivity to 14 different OmpA-specific phages, suggesting that they recognize different areas of the protein. In addition to the three clusters of mutational alterations around residues 68, 110, and 154, a site around residue 25 has been predicted to be involved in conjugation and in binding of a phage and a bacteriocin (R. Freudl, and S. T. Cole, Eur. J. Biochem, 134:497-502, 1983; G. Braun and S. T. Cole, Mol. Gen. Genet, in press). These four areas are regularly spaced, being about 40 residues apart from each other. A model is suggested in which the OmpA polypeptide repeatedly traverses the outer membrane in cross-beta structure, exposing the four areas to the outside.     

DNA sequence of genes 38 encoding a receptor-recognizing protein of bacteriophages T2, K3 and of K3 host range mutants

Genes 38, which code for a receptor-recognizing protein present at the tip of the long tail fibers, have been sequenced from phages T2, the T-even-type phage K3 and its host range mutants K3hx, K3h1 and K3h1h. The genes from phages T2 and K3 code for proteins consisting of 262 and 260 amino acid residues, respectively. Fifty amino-terminal and 25 carboxy-terminal residues are highly conserved. The amino-terminal amino acids are most likely involved in binding to the neighboring protein 37. Between residues 116 and 226 of the T2 protein and residues 116 and 223 of the K3 protein, sequences exist that are similar to sequences present in Escherichia coli outer membrane proteins and which serve as phage receptors. Most likely, all of these regions in the latter proteins are exposed on the cell surface and are part of their phage receptor areas. In the phage proteins, these sequences are flanked by stretches rich in glycine, perhaps providing an increased flexibility for the polypeptide at these sites; some "wobble" may be required during the protein 38-receptor interaction. The mutational alterations in the host range mutants were found in gene 38. In the K3hx protein, a duplication of six base-pairs caused the wild-type sequence -Gly163-Lys-Leu-Ile- to be changed to -Gly163-Lys-Leu-Lys-Leu-Ile-. In the K3h1 protein, a glutamic acid residue at position 203 was substituted by a lysine. Both alterations occurred within areas similar to outer membrane proteins. Mutant K3h1h, derived from K3h1, exhibits an extended host range as compared to K3h1. No mutational alteration, in addition to that found in K3h1, was found in g38 nor was the part of gene 37 that encodes the carboxy-terminal moiety of the protein altered. K3h1h may represent a "trigger-happy" phage. The results of this and other work show that the phage-phage receptor systems under study represent a primitive immune system.     

Host range mutants of bacteriophage Ox2 can use two different outer membrane proteins of Escherichia coli K-12 as receptors.

The Escherichia coli K-12 outer membrane protein OmpA functions as the receptor for bacteriophage Ox2. We isolated a host range mutant of this phage which was able to grow on an Ox2-resistant ompA mutant producing an altered OmpA protein. From this mutant, Ox2h5, a second-step host range mutant was recovered which formed turbid plaques on a strain completely lacking the OmpA protein. From one of these mutants, Ox2h10, a third-step host range mutant, Ox2h12, was isolated which formed clear plaques on a strain missing the OmpA protein. Ox2h10 and Ox2h12 apparently were able to use both outer membrane proteins OmpA and OmpC as receptors. Whereas there two proteins are very different with respect to primary structures and functions, the OmpC protein is very closely related to another outer membrane protein, OmpF, which was not recognized by Ox2h10 or Ox2h12. An examination of the OmpC amino acid sequence, in the regions where it differs from that of OmpF, revealed that one region shares considerable homology with a region of the OmpA protein which most likely is required for phage Ox2 receptor activity.     

Presence of DNA, encoding parts of bacteriophage tail fiber genes, in the chromosome of Escherichia coli K-12

The classical T-even bacteriophages recognize host cells with their long tail fibers. Gene products 35, 36, and 37 constitute the distal moiety of these fibers. The free ends of the tail fibers, which are formed by the CO2H terminus of gene product 37, possess the host range determinants. It was found that 4 out of 10 different strains of Escherichia coli K-12 contained regions of chromosomal DNA which hybridized with a probe consisting of genes 35, 36, and 37 of the T-even phage K3. From one strain this homologous DNA, which was associated with an EcoRI fragment of about 5 kilobases, was cloned into plasmid pUC8. Two independently recovered hybrid plasmids had undergone a peculiar rearrangement which resulted in the loss of about 3 kilobases of cloned DNA and a duplication of both the vector and the remaining chromosomal DNA. The mechanisms causing this duplication-deletion may be related to that of transposases. The cloned DNA was capable of recombination with phage T4 gene 36 and a phage T2 gene 37 amber mutant. DNA sequencing revealed the existence of regions of identity between the cloned DNA and genes 36 and 37 of phage T2. In addition, after growth of a derivative of phage K3 on a strain harboring T2 DNA, it was found that this phage contained the same parts of the T2 tail fiber genes which had been recovered from the bacterial chromosome. There appears to be little doubt that the phage had picked up this DNA from the host. The possibility is considered that a repertoire of parts of genes 36 and 37 of various T-even-type phages is present in their hosts, allowing the former to change their host ranges.     

The signal sequence of an Escherichia coli outer membrane protein can mediate translocation of a not normally secreted protein across the plasma membrane

The distal part of the long tail fibers of the Escherichia coli phage T4 consists of a dimer of protein 37. A fragment of the corresponding gene, encoding 253 amino acids, was inserted into several different sites within the cloned gene for the 325-residue outer membrane protein OmpA. In plasmid pTU T4-5 the fragment was inserted once and in pTU T4-10 tandemly twice between the codons for residues 153 and 154 of the OmpA protein. In pTU T4-22 two fragments were present, in tandem, between the codons for residues 45 and 46 of this protein. In pIN T4-6 one fragment was inserted into the ompA gene immediately following the part encoding the signal sequence. The corresponding mature proteins consist, in this order, of 605, 860, 835, and 279 amino acid residues. All precursor proteins were processed and translocated across the plasma membrane. Hence, not only can the OmpA protein serve as a vehicle for export of a nonsecretory protein, but the signal sequence alone can also mediate export of such a protein. Export of the pro-OmpA protein depends on the SecA protein. Export of the tail fiber fragment expressed from pIN T4-6 remained SecA dependent. Thus, the secA pathway in this case is chosen by the signal peptide. It is proposed that a signal peptide can mediate translocation of nonsecretory proteins as long as they are export-compatible. The inability of a signal sequence to mediate export of some proteins appears to be due to export incompatibility of the protein rather than to the absence of information, within the mature part of the polypeptide, which would be required for translocation.     

Receptor-recognizing proteins of T-even type bacteriophages. The receptor-recognizing area of proteins 37 of phages T4 TuIa and TuIb

Escherichia coli phages of the T4 family (T4, TuIa, TuIb) recognize their cellular receptors by means of a C-terminal region of protein 37; a dimer of this polypeptide (1026 residues in T4) is located at the distal part of the long tail fibers. Virions of the T2 family use protein 38 (which is attached to the free end of protein 37) for this purpose. The corresponding areas of genes 37 belonging to TuIa and TuIb were cloned and sequenced. Comparison of the deduced protein primary structures, including those of T4 and lambda Stf (Stf most likely representing a subunit of the side tail fibers of phage lambda) showed that an area of 70 to 100 residues is characterized by very variable sequences, while the sequences of the adjacent 43 to 44 C-terminal residues as well as those upstream from the variable region are highly homologous. The variable regions are flanked and interrupted seven or eight times by the motif His-x-His-y, with x and y most often being Ser or Thr; furthermore, the locations of these repeated tetrapeptides are conserved. Using hybrid phages obtained by recombination of one phage with cloned fragments of gene 37 of another, it could be shown that the area of this gene encoding receptor specificity includes the variable area. The situation is analogous to the known receptor-recognizing region of proteins 38 belonging to the T2-type family, except that the repeating sequence is of a different nature. In T4, receptor specificity is coded for by 382 base-pairs of the 3'-end of the gene, starting exactly at the variable area. It was found that T4 can use the outer membrane protein OmpC or lipopolysaccharide as receptors with the same efficiency, and it is proposed that the 70 residues of the variable part of the protein serve to bind to both ligands.     

Degrees of relatedness of T-even type E. coli phages using different or the same receptors and topology of serologically cross-reacting sites

The relatedness of a series of T-even like phages which use the Escherichia coli outer membrane protein OmpA as a receptor, and the classical phages T2, T4 and T6 has been investigated. Immunoelectron microscopy and the pattern of phage resistance in bacterial mutants revealed that: (i) phages of this morphology do not necessarily cross-react serologically; (ii) phages using different receptors may bind heterologous IgG everywhere except to the tip (comprising approximately 10% of one fiber polypeptide) of the long tail fibers; (iii) cross-reacting OmpA-specific phages may bind heterologous IgG only to the tip of these fibers: (iv) OmpA-specific phages not cross-reacting at the tip of the tail fibers use different receptor sites on the protein. Absence of cross-reactivity appears to reflect high degrees of dissimilarity. A DNA probe consisting of genes encoding the two most distal tail fiber proteins of T4 detected homologies only in DNA from phages serologically cross-reacting at this fiber. Even under conditions of low stringency, allowing the formation of stable hybrids with almost 30% base mismatch, no such homologies could be found in serologically unrelated phages. Thus, in the collection of phages examined, there are sets of very similar and very dissimilar tail fiber genes and even of such gene segments.     

Bacteriophage receptor area of outer membrane protein OmpA of Escherichia coli K-12.

A number of T-even-like bacteriophages use the outer membrane protein OmpA of Escherichia coli as a receptor. We had previously analyzed a series of ompA mutants which are resistant to such phages and which still produce the OmpA protein (R. Morona, M. Klose, and U. Henning, J. Bacteriol. 159:570-578, 1984). Mutational alterations were found near or at residues 70, 110 and 154. Based on these and other results a model was proposed showing the amino-terminal half of the 325-residue protein crossing the outer membrane repeatedly and being cell surface exposed near residues 25, 70, 110, and 154. We characterized, by DNA sequence analysis, an additional 14 independently isolated phage-resistant ompA mutants which still synthesize the protein. Six of the mutants had alterations identical to the ones described before. The other eight mutants possessed seven new alterations: Ile-24----Asn, Gly-28----Val, deletion of Glu-68, Gly-70----Cys, Ser-108----Phe, Ser-108----Pro, and Gly-154----Asp (two isolates). Only the latter alteration resulted in a conjugation-deficient phenotype. The substitutions at Ile-24 and Gly-28 confirmed the expectation that this area of the protein also participates in its phage receptor region. It is unlikely that still other such sites of the protein are involved in the binding of phage, and it appears that the phage receptor area of the protein has now been characterized completely.     

Role of lipopolysaccharide in assembly of Escherichia coli outer membrane proteins OmpA, OmpC, and OmpF.

Selection was performed for resistance to a phage, Ox2, specific for the Escherichia coli outer membrane protein OmpA, under conditions which excluded recovery of ompA mutants. All mutants analyzed produced normal quantities of OmpA, which was also normally assembled in the outer membrane. They had become essentially resistant to OmpC and OmpF-specific phages and synthesized these outer membrane porins at much reduced rates. The inhibition of synthesis acted at the level of translation. This was due to the presence of lipopolysaccharides (LPS) with defective core oligosaccharides. Cerulenin blocks fatty acid synthesis and therefore that of LPS. It also inhibits synthesis of OmpC and OmpF but not of OmpA (C. Bocquet-Pagès, C. Lazdunski, and A. Lazdunski, Eur. J. Biochem. 118:105-111, 1981). In the presence of the antibiotic, OmpA synthesis and membrane incorporation remained unaffected at a time when OmpC and OmpF synthesis had almost ceased. The similarity of these results with those obtained with the mutants suggests that normal porin synthesis is not only interfered with by production of mutant LPS but also requires de novo synthesis of LPS. Since synthesis and assembly of OmpA into the outer membrane was not affected in the mutants or in the presence of cerulenin, association of this protein with LPS appears to occur with outer membrane-located LPS.     

Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7

Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages. To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage. Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E. coli K12 cells. The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E. coli O157:H7 and related species. On the other hand, T2ppD1 could not infect E. coli K12, the original host of T2, or its derivatives. The host range of T2ppD1 was the same as that of PP01. Infection of T2ppD1 produced turbid plaques on a lawn of E. coli O157:H7 cells. The binding affinity of T2ppD1 to E. coli O157:H7 was weaker than that of PP01. The adsorption rate constant (ka) of T2ppD1 (0.17 x 10(-9)(ml CFU(-1) min(-1)) was almost 1/6 that of PP01 (1.10 x 10(-9)(ml CFU(-1) min(-1))). In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage.     

Mutants defective in the 33K outer membrane protein of Salmonella typhimurium.

Salmonella typhimurium LT2 lines, if phenotypically rough, are fully sensitive to bacteriocin 4-59, produced by Salmonella canastel strain SL1712. Bacteriocin-resistant mutants fell into three classes. Those resistant to phage ES18 and to albomycin proved to be mutants of class chr (equivalent to tonB of Escherichia coli); these mutants still adsorb the bacteriocin and so are classified as tolerant. Another class of (incompletely) tolerant mutants was resistant to phage PH51; their envelope fractions lacked the band corresponding to outer membrane protein 34K, known to serve for adsorption of phage PH51. A third class of mutants, which did not adsorb the bacteriocin, was unaltered in sensitivity to phages. Their envelopes lacked the 33K band, indicating absence of the outer membrane protein 33K, considered to correspond to outer membrane protein II* of E. coli, which in that species is determined at locus ompA (formerly tolG or con). Phage P22 HT105/1 cotransduced the 33K S. typhimurium gene (to be called ompA, to accord with E. coli usage) with pyrD+ at about 30% frequency when the donor allele was ompA+ or one ompA, but at only 3 to 11% when the donor allele was another ompA. When the donor carried either of two long deletions of the put (proline utilization) operon, phage P22 HT105/1 cotransduced put (and ompA+) with pyrD+ at low frequency. The cotransduction data indicate that ompA of S. typhimurium is located between pyrD and put, nearer the former. This corresponds to the map position of ompA in E. coli K-12.     

Phage receptors of Escherichia coli. Basic components of the outer membrane of E. coli as phage receptors

Major outer membrane components which determine the structure and the barrier function of membrane Gram-negative bacteria are receptors for many bacteriophages. LPS--the major component of the outer membrane of Enterobacteria can be used by some phages with wide host range specificity. The other component of the outer membrane frequently include phage receptor component is OmpA protein. OmpA protein different areas can be used as receptors for different phages T--even group. A large group of phage receptors compose porin proteins, which are discovered in 32 species of bacteria. The synthesis of major porin proteins, which a receptor for several phages, are regulated by sufficiently complex system of some genes. These genes are sensitive to the changes of environment.     

Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells

Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. Klebsiella pneumoniae is a capsulated gram-negative pathogen. It is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by a lack of an early inflammatory response. Data from our laboratory indicate that K. pneumoniae CPS helps to suppress the host inflammatory response. However, it is unknown whether K. pneumoniae employs additional factors to modulate host inflammatory responses. Here, we report that K. pneumoniae OmpA is important for immune evasion in vitro and in vivo. Infection of A549 and normal human bronchial cells with 52OmpA2, an ompA mutant, increased the levels of IL-8. 52145-Δwca(K2)ompA, which does not express CPS and ompA, induced the highest levels of IL-8. Both mutants could be complemented. In vivo, 52OmpA2 induced higher levels of tnfα, kc, and il6 than the wild type. ompA mutants activated NF-κB, and the phosphorylation of p38, p44/42, and JNK MAPKs and IL-8 induction was via NF-κB-dependent and p38- and p44/42-dependent pathways. 52OmpA2 engaged TLR2 and -4 to activate NF-κB, whereas 52145-Δwca(K2)ompA activated not only TLR2 and TLR4 but also NOD1. Finally, we demonstrate that the ompA mutant is attenuated in the pneumonia mouse model. The results of this study indicate that K. pneumoniae OmpA contributes to attenuate airway cell responses. This may facilitate pathogen survival in the hostile environment of the lung.     

A component of the side tail fiber of Escherichia coli bacteriophage lambda can functionally replace the receptor-recognizing part of a long tail fiber protein of the unrelated bacteriophage T4.

The distal part of the long tail fiber of Escherichia coli bacteriophage T4 consists of a dimer of protein 37. Dimerization requires the catalytic action of protein 38, which is encoded by T4 and is not present in the virion. It had previously been shown that gene tfa of the otherwise entirely unrelated phage lambda can functionally replace gene 38. Open reading frame (ORF) 314, which encodes a protein that exhibits homology to a COOH-terminal area of protein 37, is located immediately upstream of tfa. The gene was cloned and expressed in E. coli. An antiserum against the corresponding polypeptide showed that it was present in phage lambda. The serum also reacted with the long tail fibers of phage T4 near their free ends. An area of the gene encoding a COOH-terminal region of ORF 314 was recombined, together with tfa, into the genome of T4, thus replacing gene 38 and a part of gene 37 that codes for a COOH-terminal part of protein 37. Such T4-lambda hybrids, unlike T4, required the presence of outer membrane protein OmpC for infection of E. coli B. An ompC missense mutant of E. coli K-12, which was still sensitive to T4, was resistant to these hybrids. We conclude that the ORF 314 protein represents a subunit of the side tail fibers of phage lambda which probably recognize the OmpC protein. ORF 314 was designated stf (side tail fiber). The results also offer an explanation for the very unusual fact that, despite identical genomic organizations, T4 and T2 produce totally different proteins 38. An ancestor of T4 from the T2 lineage may have picked up tfa and stf from a lambdoid phase, thus possibly demonstrating horizontal gene transfer between unrelated phage species.