The cell wall receptor for bacteriophage Mu G(−) in Erwinia and Escherichia coli C

Abstract Adsorption of bacteriophage Mu with its invertible DNA segment in the G(−) orientation requires a terminal glucose residue for binding to the core lipopolysaccharide (LPS) of Gram-negative bacteria. Analysis of a Mu-resistant mutant shows that the receptor for Mu G(−) in Erwinia B374 is a Glc-β1,6-Glc disaccharide. A spontaneously occurring host-range mutant, Mu G(−)h101, grows on Escherichia coli C. The loss of the terminal β1,3-linked glucose from the LPS of E. coli C leads to resistance to the phage Mu. These mutants are also resistant to phage P1 and D108 which have largely homologous G segments. This shows that Mu G(+) and G(−) phage particles differ with respect to their cell-wall receptors in the type of glycosidic linkage of a terminal glucose residue: α1, 2 for G(+) and β1,6 for G(−).     

Cell wall receptor for bacteriophage Mu G(+).

The invertible G segment in phage Mu DNA controls the host range of the phage. Depending on the orientation of the G segment, two types of phage particles, G(+) and G(-), are produced which recognize different cell surface receptors. The receptor for Mu G(+) was located in the lipopolysaccharide (LPS) of gram-negative bacteria. The analysis of different LPS core types and of mutants that were made resistant to Mu G(+) shows that the primary receptor site on Escherichia coli K-12 lies in the GlcNAc beta 1 . . . 6Glc alpha 1-2Glc alpha 1-part at the outer end of the LPS. Mu shares this receptor site in E. coli K-12 with the unrelated single-stranded DNA phage St-1. Phage D108, which is related to Mu, and phages P1 and P7, which are unrelated to Mu but contain a homologous invertible DNA segment, have different receptor requirements. Since they also bind to terminal glucose in a different configuration, they adsorb to and infect E. coli K-12 strains with an incomplete LPS core.     

G inversion in bacteriophage Mu DNA is stimulated by a site within the invertase gene and a host factor

The Gin function of bacteriophage Mu catalyzes inversion of the G DNA segment, thus switching the host range of Mu phage particles. This site-specific recombination event takes place between inverted repeat sequences (IR) that border the G segment. Sequences in the Mu beta region extending approximately from position 118 to 178 are essential for efficient inversion. In cis this region, termed sis, stimulates inversion about 15-fold. Neither the relative orientation of sis with respect to the IR sequences nor the distance to IR substantially influences the stimulatory effect. For full activity purified Gin protein must be supplemented with crude host factor from E. coli K12. We suggest that, in addition to Gin, a DNA-binding host protein is required for efficient G inversion.     

Isolation and characteristics of mutation pfm affecting the penetration of phage Mu into Escherichia coli K-12 cells

A mutant of Escherichia coli K-12 strain with the destroyed process of establishment of lysogenic state for phage Mu in the course of zygotic induction has been obtained. The mutation revealed, designated pfm (penetration factor for Mu), interferes with adsorption of phage Mu to the surface of E. coli K-12 cells. On the basis of data obtained, there is every reason to believe that the phage Mu DNA connection with the membrane components of the bacterial cell provides optimizing condition for the primary integrative transposition of phage Mu at the stage of Mu DNA introduction into the cell.     

Purification of the Gin recombination protein of Escherichia coli phage Mu and its host factor

Inversion of the G-segment of Escherichia coli phage Mu was studied in vitro. The reaction requires the Gin recombination protein, which was purified to near homogeneity from overproducing cells. Upon purification the protein lost activity, which was restored by addition of an extract from uninfected E. coli cells. The stimulatory host factor is a small heat-stable protein and was purified from E. coli cells. Full recombination required both proteins, but Gin alone promoted some recombination by itself, particularly at high concentrations. Relaxation of negative supercoils and recombination of a substrate with two recombination sites in an inverted orientation both have the same specificity for Gin and the host factor. The Gin-associated topoisomerase activity appears tightly coupled to its recombination activity.     

Adsorptive capacity of bacteriophage Mu on Escherichia coli K-12 strains with deletions in the attlambda region]

Escherichia coli strains with deletions in att lambda region were obtained. The comparison of the extent of deletions with the sensitivity of the corresponding mutant clones to phage Mu showed that the gene controlling the sensitivity of E. coli K-12 to the phage Mu is located in nad A-gal region of the bacterial chromosome. It is shown that the resistance of E. coli strains which had lost the region of bacterial chromosome between nad A gene and genes of gal-operon have adsorption character. Deletion of the nad A-gal region does not affect the adsorption of other phages (lambda, P1 and T4). Thus, the gene, located in this region, is responsible for the specific adsorption of the phage Mu.     

A Mu gin complementing function and an invertible DNA region in Escherichia coli K-12 are situated on the genetic element e14

The Gin product catalyzes an inversion of 3,000 base pairs of DNA in the genome of bacteriophage Mu. The orientation of the invertible of G-region determines the host range of the phage. Gin- mutants are complemented by a host function in strain HB101 and several other Escherichia coli K-12 strains. At least three clones in the E. coli gene bank described previously (L. Clarke and J. Carbon, Cell 9:91-99, 1976) contained the gin complementing function. This function, which we named pin, catalyzes an inversion of 1,800 base pairs in the adjacent DNA. The invertible region, named the P-region, together with pin, was further subcloned on pBR322. Conjugation and transduction experiments mapped the pin gene between the genes purB and fabD near position 25 on the E. coli chromosome. Also situated in this region is e14, a cryptic, UV- excisable , genetic element (A. Greener and C.W. Hill, J. Bacteriol . 144:312-321, 1980). We demonstrated that pin and the P-region are part of e 14. The e 14 element was cloned on pBR322 by genetic manipulation techniques in vivo. It has the properties of a defective prophage containing integration and excision functions and a SOS-sensitive repressor.     

Cloning of the ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) structural genes from Salmonella typhimurium LT2.

The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the SalI site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in lambda 1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the SalI site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coli K-12 RR1(pPL301) were increased 6- to 7-fold and 13- to 15-fold, respectively. Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.     

Coding theory based models for protein translation initiation in prokaryotic organisms

Our research explores the feasibility of using communication theory, error control (EC) coding theory specifically, for quantitatively modeling the protein translation initiation mechanism. The messenger RNA (mRNA) of Escherichia coli K-12 is modeled as a noisy (errored), encoded signal and the ribosome as a minimum Hamming distance decoder, where the 16S ribosomal RNA (rRNA) serves as a template for generating a set of valid codewords (the codebook). We tested the E. coli based coding models on 5' untranslated leader sequences of prokaryotic organisms of varying taxonomical relation to E. coli including: Salmonella typhimurium LT2, Bacillus subtilis, and Staphylococcus aureus Mu50. The model identified regions on the 5' untranslated leader where the minimum Hamming distance values of translated mRNA sub-sequences and non-translated genomic sequences differ the most. These regions correspond to the Shine-Dalgarno domain and the non-random domain. Applying the EC coding-based models to B. subtilis, and S. aureus Mu50 yielded results similar to those for E. coli K-12. Contrary to our expectations, the behavior of S. typhimurium LT2, the more taxonomically related to E. coli, resembled that of the non-translated sequence group.     

A gene for DNA invertase and an invertible DNA in Escherichia coli K-12

An assay system for the pin gene function, which suppresses the vh2 mutation of Salmonella, was developed and used to show that most strains of Escherichia coli K-12 are Pin+, whereas all the strains of E. coli C examined are Pin-. An E. coli host strain was constructed and used for detection of DNA fragments carrying the E. coli K-12 pin gene cloned in the plasmid vector pBR322. Restriction analysis of the cloned fragments showed that the invertible DNA (designated P region) is adjacent to the pin gene and that its inversion is mediated by the pin gene product. The pin gene was found to be functionally homologous to the gin gene of Mu phage and the cin gene of P1 phage. The P region most probably resides within the cryptic prophage e14, and the Pin- phenotype is likely to be associated with the loss of e14.     

Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion in vitro

Inversion of the G segment in bacteriophage Mu DNA occurs by a site-specific recombination event and determines the host specificity of Mu phage particles produced. Inversion is mediated by a Mu function (Gin). The gin gene has been placed under control of the inducible λ pL promoter and a synthetic Shine-Dalgarno linker upstream of the initiation codon. The Gin protein content in induced cells is boosted to ˜10% of total protein. Partially purified extracts from overproducing strains promote efficient inversion of the G DNA segment in vitro which is visualized by agarose gel electrophoresis of the substrate DNA after cutting with appropriate restriction endonucleases. The in vitro reaction requires Mg²⁺, a super-coiled DNA substrate and occurs in the absence of exogenous ATP. Inversion from the G(+) to the G(−) orientation is as efficient as the switch from G(−) to G(+).     

Cloning of genes from members of the family Enterobacteriaceae with mini-Mu bacteriophage containing plasmid replicons.

An in vivo cloning system that uses derivatives of the Escherichia coli bacteriophage Mu with plasmid replicons has been extended to five different species of the family Enterobacteriaceae. Mu and these mini-Mu replicon elements were introduced into strains of E. coli, Shigella flexneri, Salmonella typhimurium, Citrobacter freundii, and Proteus mirabilis by infection, by transformation, or by conjugation with newly constructed broad-host-range plasmids containing insertions of these elements. Lysates from these cells, lysogenic for Mu and mini-Mu elements, were used to infect sensitive recipient strains of E. coli, S. typhimurium, and C. freundii. Drug-resistant transductants had mini-Mu replicon elements with inserts of different DNA sequences. All of the lysogens made could be induced to yield high phage titers, including those coming from strains that were resistant to Mu and Mu derivatives. Clones of 10 particular genes were isolated by their ability to complement specific mutations in the recipient strains, even in the presence of the E. coli K-12 restriction system. Some of the mini-Mu replicon elements used contained lac gene fusing segments and resulted in fusions of the lac operon to control regions in the cloned sequences.     

Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli.

The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.     

Transposition of the deo operon structural genes in Escherichia coli K-12 to plasmid RP4 using bacteriophage mu]

Transposition of the structural genes of the deo operon of Escherichia coli K-12 into plasmid RP4 by means of temperate bacteriophage Mu was carried out. Some variants of composite RP4-deo-Mu plasmids were obtained and the expression of the deo genes integrated into the RP4 plasmid genome was studied. It was shown that the expression of these genes remains under the control of the chromosomal regulatory genes (deoR and cytR); although the activity of thymidine phosphorilase in the strain E. coli which contains hybrid plasmid is 4-6 fold greater than that in strains of E. coli with chromosomal localization of the deo operon.     

Evidence for effect of random genetic drift on G+C content after lateral transfer of fucose pathway genes to Escherichia coli K-12

The cps cluster of Escherichia coli K-12 comprises genes involved in synthesis of capsular polysaccharide colanic acid. Part of the E. coli K-12 cps region has been cloned and sequenced and compared to its Salmonella enterica LT2 counterpart. The cps genes from the two organisms are homologous; in the case of the LT2 genes, with G+C content of 0.61 and codons characteristic of high G+C species, it seems clear that they have been acquired relatively recently by lateral transfer from a high G+C species. The K-12 form of these cps genes is closely related to those of LT2 so must derive from the same high G+C species, but it appears to have transferred much earlier such that random genetic drift has brought P3 (the corrected G+C content of codon base 3) down from 0.77 to 0.64, more than halfway to the E. coli average of 0.57. We estimate, using an equation developed by Sueoka, that the lateral transfer to E. coli took place approximately 45 million years ago. This is the first report we are aware of demonstrating the expected adjustment of P3 after lateral transfer between species with different G+C content DNA.      

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster.

Escherichia coli K-12 has long been known not to produce an O antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of O antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rfb (O antigen) genes intact which synthesized a variant of O antigen O16, giving cross-reaction with anti-O17 antibody. We determined the structure of this O antigen to be -->2)-beta-D-Galf-(1-->6)-alpha-D-Glcp- (1-->3)-alpha-L-Rhap-(1-->3)-alpha-D-GlcpNAc-(1-->, with an O-acetyl group on C-2 of the rhamnose and a side chain alpha-D-Glcp on C-6 of GlcNAc. O antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rfb (O antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rfb region of Escherichia coli K-12 and those of E. coli O4 and E. coli Flexneri. All K-12 rfb genes were of low G+C content for E. coli. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rfb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.     

Infection of Salmonella typhimurium with coliphage Mu d1 (Apr lac): construction of pyr::lac gene fusions.

A procedure was developed for introducing the coliphage Mu d1 (Apr lac) into Salmonella typhimurium in order to construct gene fusions that place the structural genes of the lac operon under the control of the promoter-regulatory region of other genes. To introduce Mu d1 from Escherichia coli K-12 into S. typhimurium, which is normally not a host for Mu, we first constructed an E. coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid helper phage (MuhP1) that confers the P1 host range. A lysate prepared from this strain was used to infect a P1-sensitive (i.e., galE), restriction-deficient, modification-proficient strain of S. typhimurium, and a double lysogen carrying Mu d1 and MuhP1 was isolated. Induction of the latter strain produced lysates capable of infecting and generating gene fusions in P1-sensitive strains of S. typhimurium. In this paper we describe the construction of pyr::lac fusions by this technique.     

A second prepilin peptidase gene in Escherichia coli K-12

Escherichia coli K-12 strains grown at 37°C or 42°C, but not at 30°C, process the precursors of the Neisseria gonorrhoeae type IV pilin PilE and the Klebsiella oxytoca type IV pseudopilin PulG in a manner reminiscent of the prepilin peptidase-dependent processing of these proteins that occurs in these bacteria. Processing of prePulG in Escherichia coli requires a glycine at position −1, as does processing by the cognate prepilin peptidase (PulO), and is unaffected by mutations that inactivate several non-specific proteases. These data suggested that E . coli K-12 has a functional prepilin peptidase, despite the fact that it does not itself appear to express either type IV pilin or pseudopilin genes under the conditions that allow prePilE and prePulG processing. The E . coli K-12 genome contains two genes encoding proteins with significant sequence similarity to prepilin peptidases: gspO at minute 74.5 and pppA (f310c) at minute 67 on the genetic map. We have previously obtained evidence that gspO encodes an active enzyme but is not transcribed. pppA was cloned and shown to code for a functional prepilin peptidase capable of processing typical prepilin peptidase substrates. Inactivation of pppA eliminated the endogenous, thermoinducible prepilin peptidase activity. PppA was able to replace PulO prepilin peptidase in a pullulanase secretion system reconstituted in E . coli when expressed from high-copy-number plasmids but not when present in a single chromosomal copy. The analysis of pppA–lacZ fusions indicated that pppA expression was very low and regulated by the growth temperature at the level of translation, in agreement with the observed temperature dependence of PppA activity. Polymerase chain reaction and Southern hybridization analyses revealed the presence of the pppA gene in 12 out of 15 E . coli isolates.