Behavior of coliphage lambda in hybrids between Escherichia coli and Salmonella

Salmonella typhosa hybrids able to adsorb lambda were obtained by mating S. typhosa recipients with Escherichia coli K-12 donors. After adsorption of wild-type lambda to these S. typhosa hybrids, no plaques or infective centers could be detected. E. coli K-12 gal(+) genes carried by the defective phage lambdadg were transduced to S. typhosa hybrids with HFT lysates derived from E. coli heterogenotes. The lysogenic state which resulted in the S. typhosa hybrids after gal(+) transduction differed from that of E. coli. Ability to produce lambda, initially present, was permanently segregated by transductants of the S. typhosa hybrid. S. typhosa lysogens did not lyse upon treatment for phage induction with mitomycin C, ultraviolet light, or heat in the case of thermoinducible lambda. A further difference in the behavior of lambda in Salmonella hybrids was the absence of zygotic induction of the prophage when transferred from E. coli K-12 donors to S. typhosa. A new lambda mutant class, capable of forming plaques on S. typhosa hybrids refractory to wild-type lambda, was isolated at low frequency by plating lambda on S. typhosa hybrid WR4254. Such mutants have been designated as lambdasx, and a mutant allele of lambdasx was located between the P and Q genes of the lambda chromosome. Plaques were formed also on the S. typhosa hybrid host with a series of lambda(i21) hybrid phages which contain the N gene of phage 21. The significance of these results in terms of Salmonella species as hosts for lambda is discussed.     

Specialized Transducing Phages Derived from Salmonella Phage P22

Salmonella phage P22 has been used in the construction of three sorts of specialized transducing phage: P22 proAB, P22 proABlac and P22 argF. The bacterial genes carried are derived from E. coli K12. Since E. coli and Salmonella chromosomes recombine very poorly, E. coli genes cannot be transduced into Salmonella recipients by P22's generalized transduction mechanism. Therefore, stable inheritance of E. coli material provides a means of detecting specialized transduction. Formation of these phages was possible because the P22 prophage recognizes an attachment site in the E. coli F' prolac episome. Salmonella strains carrying the F' prolac episome can be lysogenized by P22 so as to leave the prophage inserted into the E. coli material of the F' factor. Improper prophage excision can then lead to formation of P22 specialized phages carrying E. coli genetic material.     

A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.     

Influence of O Side Chains on the Attachment of the Felix O-1 Bacteriophage to Salmonella Bacteria

The adsorption rate constant (ARC) of the Felix O-1 (FO) bacteriophage to sensitive Salmonella strains was used to determine the effect of variations in surface antigens on phage attachment. The N-acetylglucosamine of the common-core polysaccharide of the Salmonella lipopolysaccharide (LPS) was found to be an essential part of the receptor for the FO phage in conformation with earlier reports. It was found that (i) the ARC was low for strains having O side chains containing two or three non core monosaccharides, (ii) the ARC varied when the O side chain contained no, or only one, noncore monosaccharide, (iii) the ARC was high when the O side chain contained only one repeating unit, and (iv) the ARC was high to mutants of chemotype Ra in which the N-acetylglucosamine was the terminal sugar of the LPS. Since a good correlation was found between the ARC of the FO phage and the phage-inactivating capacity of phenol water-extracted LPS, the results suggest that only the structure and composition of the LPS determines the adsorption rate of the FO phage. The phage-inactivating capacity of LPS from the Ra mutants increased in parallel with higher glucosamine contents in the core polysaccharide. In smooth strains having long and numerous O side chains, the access of the FO phage to its receptor is probably blocked by the presence of the side chains, whereas short and numerous side chains or T1 side chains do not interfere with the FO attachment.     

Genetic determination of the O antigens of Salmonella groups B (4,5,12) and C1(6,7)

M?kel?, P. Helena (University of Helsinki, Helsinki, Finland). Genetic determination of the O antigens of Salmonella groups B (4,5,12) and C(1) (6,7). J. Bacteriol. 91:1115-1125. 1966.-The genetic determination of the O antigens of Salmonella was studied by Hfr or F' crosses between strains of groups B (antigens 4,5,12) and C(1) (antigens 6,7). The main genetic determinants of the specificities 4 of group B and 7 of group C(1) behaved as alleles of one locus, called O or O-4/7. This is probably identical with O-4/9, responsible for the serological difference between groups B and D, and with the "rough" locus rouB. At least parts of the antigens 12 of group B and 6(2) of group C are determined at the same locus. The gene O-5 is closely linked to O-4/7, both mapping in the approximately 2 minutes distance between his and metG in the order his - O-4/7 - O-5 - metG. In crosses of group B donors with group C(1) recipients, a serologically new type, called semirough (SR), appeared in most recombinants that had inherited the O-4 allele of the group B donor. These SR forms are serologically intermediate between smooth and rough forms, showing poor stability in saline but possessing the specificities 4 and 12 and (some of them) 5. On the basis of previous biochemical studies, the hypothesis has been put forward that the side chains of their lipopolysaccharide are much shorter than normal group B side chains, probably containing only one repeating unit per side chain. A gene SR-4 responsible for the elongation of group B side chains beyond the first repeating unit was mapped between gal and try, group B and D bacteria being Sr-4(+), and group C being SR-4(-).     

Transduction with Integration-Defective Mutants of Salmonella typhimurium Bacteriophage KB1

Phage KB1 has gained use as a generalized transducing phage for Salmonella typhimurium, including strains resistant to phage P22. Integration-defective mutants of KB1 have now been isolated; one of these, int-1, is recommended for transduction when nonlysogenic recombinants are desired.     

Characterization of SE1, a new general transducing phage of Salmonella typhimurium

A transducing phage, SE1, which is able to infect Salmonella typhimurium was isolated from a Salmonella enteritidis strain. SE1 is a temperate phage which is heteroimmune with respect to phages P22, L, KB1 and ES18. It is similar in morphology and size to phages P22, L and KB1 and is serologically related to phages P22 and L but not to KB1. Efficiencies of generalized transduction effected by phage SE1 are similar to those for P22HT (int7), a mutant which mediates a high frequency of chromosomal gene transduction. The lengths of chromosomal DNA transduced by SE1 and P22HT (int7) are similar. Furthermore, the SE1 prophage does not exclude the transducing particles from cells it has lysogenized; consequently it is possible to use both SE1 lysogens and non-lysogenic strains as recipients in SE1-mediated transduction experiments, and obtain similar transduction efficiencies. However, the SE1 prophage gives rise to a lysogenic conversion that decreases the rate of adsorption of SE1 and L phages by about 50%, but does not affect adsorption of P22. Altogether these results suggest that phage SE1 may be a useful tool in the genetic manipulation of S. typhimurium.     

Role of lipopolysaccharide in the receptor function for bacteriophage Ox2

Abstract The sensitivity of Salmonella typhimurium as well as of Escherichia coli to the bacteriophage Ox2 was found to require, in addition to the OmpA protein, a certain type of rough LPS. Heptoseless mutants were resistant and unable to adsorb the phage. Mutants with less defective LPS chemotype were sensitive and could, except the second most defective chemotypes, adsorb Ox2. However, isolated LPS-free OmpA protein could bind the phage, and this binding could not be increased by adding LPS.     

Factors that affect transduction in microorganisms]

Data from literature concerning general and specialized transduction in microorganisms are given in the paper. The process of exogenic DNA penetration to the cells of bacteria and participation of protein products of separate phage genes in this process are described. The so-called E-proteins in a set with DNA penetrate through a cell membrane. In phage P22 they are protein products of phage genes 7, 16, 20. In P22 mutants with an altered transducing frequencies (HFT and LFT) the due functions are also coded by the phage genes. It is shown that the process of DNA packing in phages P22, phi 80, lambda and others is genetically determined. The gene transfer frequency depends on UV radiation and the very nature of transducing phages itself. In virulent phages the UV radiation up to inactivation level 95-99% evokes a decrease of their "killer" ability, which is accompanied by an increase of survivability of the formed transductants and, as a result, by enhancement of the transduction transfer frequency. An important role of the transduction analysis for fine mapping of a genome of microorganisms and its significance for practice are shown. A mathematical analysis of the data on cotransduction of linkage markers is presented as such that may be used when determining the value of transduced fragment of a chromosome.     

Conditionally transposition-defective derivative of Mu d1(Amp Lac).

A Mu d1 derivative is described which is useful for genetic manipulation of Mu-lac fusion insertions. A double mutant of the specialized transducing phage Mu d1(Amp Lac c62ts) was isolated which is conditionally defective in transposition ability. The Mu d1 derivative, designated Mu d1-8(Tpn[Am] Amp Lac c62ts), carries mutations which virtually eliminate transposition in strains lacking an amber suppressor. In such strains, the Mu d1-8 prophage behaves like a standard transposon. It can be moved from one strain of Salmonella typhimurium to another by the general transducing phage P22 with almost 100% inheritance of the donor insertion mutation. When introduced into a recipient carrying supD, supE, or supF, 89 to 94% of the Ampr transductants were transpositions of the donor Mu d1-8, from the transduced fragment into new sites. The stability of Mu d1-8 in a wild-type, suppressor-free background was sufficient to permit use of the fusion to select constitutive mutations without prior isolation of deletions to stabilize the fusion. Fusion strains could be grown at elevated temperature without induction of the Mu d prophage. The transposition defect of Mu d1-8 was corrected by a plasmid carrying the Mu A and B genes.     

STAPHYLOCOCCAL TRANSDUCING PARTICLE.

Dowell, C. E. (The University of Texas, Dallas) and E. D. Rosenblum. Staphylococcal transducing particle. J. Bacteriol. 84:1076-1079. 1962.-When novobiocin-resistant transductants were isolated under conditions that permitted superinfection, almost all the clones were lysogenic for the transducing phage. If superinfection was prevented, then the transductants isolated were nonlysogenic, suggesting the defective nature of the transducing particle. It was noted that the transducing and plaque-forming particles showed no appreciable difference in buoyant density. No difference was found in transduction rates when either sensitive or lysogenic cells were used as recipients. Transduction rates as high as one transductant per 7 x 10(4) phage particles were obtained for novobiocin resistance.     

Bacteriophage attachment sites, serological specificity, and chemical composition of the lipopolysaccharides of semirough and rough mutants of Salmonella typhimurium

Extracted lipopolysaccharides (LPS) from one smooth, one semirough, and five rough mutants of Salmonella typhimurium LT2 or LT7, for which the chemical structure of the polysaccharide chain had been elucidated by using methylation analysis, were characterized with passive hemagglutination inhibition and phage inactivation experiments. Each addition of a sugar residue to a LPS from chemotype Rc was reflected in changed serological reactivity and phage-inhibiting activity of a collection of bacteriophages of the isolated LPS. Thus, certain criteria can be established for a classification of rough mutants of S. typhimurium. The observation that the serological RII specificity corresponds to a completed common core polysaccharide was verified. The serological RI specificity was found in LPS with terminal d-galactose I residues. One of the mutants, SL733, yielded a LPS which cross-reacted with anti-O5 factor serum although the polysaccharide was virtually free from contaminating O-specific material. The O5 reactivity was destroyed by alkaline treatment of SL733 LPS. The smooth- and rough-specific Felix O-l (FO) and the rough-specific 6SR and Br2 phages were shown to have their receptors in the LPS. There was a good correlation between the adsorption rate constant to whole cells and the phage inhibiting activity of isolated LPS suggesting that the LPS exert the major influence on the attachment of these phages to the bacteria. The polysaccharide structures in the LPS necessary for attachment of the 6SR and Br2 phages were defined. It was found that measuring the phage-inhibiting properties of isolated LPS as PhI(50) (LPS concentration required to inactivate 50% of the phages under defined conditions) was a more sensitive method for a characterization of the LPS than the serological and chemical assays used.     

Effects of the Recipient Strain and Ultraviolet Irradiation on Transduction Kinetics of the Penicillinase Plasmid of Staphylococcus aureus

When the penicillinase plasmid of Staphylococcus aureus PS 81(P(81))(T(81)) was transferred to its cured derivative of PS 81(N(P))(T(81)), there was a fivefold increase in the transduction frequency of penicillinase plasmid markers after ultraviolet (UV) irradiation of the phage instead of the expected decrease typical for plasmid-borne markers. These results were independent of the transducing phage, the donor, and the method of curing the recipient and were also obtained with a cured derivative of PS 80(PI(80)). With PS 52, a naturally occurring penicillin-sensitive strain, and a cured transductant of PS 52 as the recipients, typical plasmid kinetics were observed. The plasmid location of penicillinase plasmid markers in transductants was confirmed by their instability in ethidium bromide (EB). In a cross between isogenic plasmids (PI(258)penZ cad x PI(258)penI asa ero), transductants were doubly selected for cadmium and erythromycin resistances. There was a twofold increase in transduction frequency after UV irradiation of the transducing phage and an increase in the proportion of recombinant type transductants. CsCl-EB density centrifugation revealed that plasmid deoxyribonucleic acid (DNA) was present in PS 81(P(81))(N(T)) and its cured derivative [PS 81(N(P))(N(T))], but not in PS 52. Sucrose gradient analysis of plasmid DNA showed that the penicillinase plasmid of PS 81(P(81))(N(T)) was larger than the plasmid in its cured derivative. Thus, the cured derivative contains plasmid DNA which appears to recombine with the incoming plasmid, causing the rise in transduction frequency noted after UV irradiation of transducing 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.     

Mapping of the genetic determinant controlling Salmonella K-antigen synthesis. II. Nonmotile mutants of Salmonella typhimurium

According to the preliminary data, S. typhimurium K-antigen is located in the area of minutes 40-44 on the Salmonella chromosome map. The formation of nonmotile mutants from motile Salmonella strains was induced by the action of nitrosoguanidine. Two main groups of mutants differing in their reaction of agglutination with H- and K-antisera were obtained: Mot-H-K- (motA or motB mutants) and Mot-H-K- (H1- or fla- mutants). The transduction transfer of the sign of motility by phage P22HT to H-K- mutants and to H1- and flaE- mutants led to the restoration of agglutination ability with respect to H- and K-antisera in all Mot+ transductants under study simultaneously. The restoration of H+K+ phenotype was also observed in spontaneous motile revertants obtained from H-K- mutants. Thus, the gene controlling the synthesis of K-antigen in Salmonellae was shown to be incorporated into the Fla operon, the regulatory system of the operon controlling the expression of this gene.     

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: high frequency of aberrant prophage excision.

The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanisms. Based on the properties of the DNA packaging mechanism of phage P22 a model for the generation of various types of specialized transducing particles is presented that suggests generation of substantial numbers of specialized transducing genomes which are heterogeneous but only some of which have terminally redundant ends. The primary attachment site, ataA, for phage P22 in S. typhimurium is located between the genes proA,B and supQ newD. (The newD gene is a substitute gene for the leuD gene, restoring leucine prototrophy of leuD mutant strains.) The proA,B and supQ newD genes are very closely linked and thus cotransducible by generalized transducing particles. Specialized transducing particles can carry either proA,B or supQ newD but not both simultaneously, and thus cannot give rise to cotransduction of the proA,B and supQ newD genes. This difference is used to calculate the frequency of generalized and specialized transducing particles from the observed cotransduction frequency in phage lysates. By this method, very high frequencies of supQ newD (10(-2)/PFU)- and proA,B (10(-3)/PFU)-specialized transducing particles were detected in lysates produced by induction of lysogenic strains. These transducing particles most of which would have been produced by independent aberrant excision events (which include in situ packaging), were of various types.     

Heterogeneity in expression of lipopolysaccharide by strains of Salmonella enterica serotype Typhimurium definitive phage type 104 and related phage types

AIMS: To investigate lipopolysaccharide (LPS) expression in Salmonella enterica serotype Typhimurium definitive phage type 104 (Salmonella Typhimurium DT104) and related phage types. METHODS AND RESULTS: Isolates were examined for the expression of LPS by SDS-PAGE and silver staining and subtyped by Pulsed Field Gel Electrophoresis (PFGE). The 100 isolates expressed one of two LPS profiles designated A (72%) and B (28%). LPS profiling was able to discriminate between isolates of identical PFGE type. Among 10 groups of outbreak isolates examined, each group was of a single LPS profile: A, 8/10 and B, 2/10. All 10 outbreaks were identical by PFGE analysis. CONCLUSIONS: Isolates of Salmonella Typhimurium DT104 and related phage types expressed one of two distinct LPS profiles. The two LPS profiles appear similar but shifted and in phase with one another, suggesting that the heterogeneity is due to changes in the LPS core region rather than among the repeating oligosaccharide units of the long-chain LPS. SIGNIFICANCE AND IMPACT OF THE SUTDY: LPS profiling provides a useful adjunct to PFGE and other molecular methods for the subtyping of this group of bacteria in epidemiological investigations.     

Resistance of Salmonella typhimurium mutants to galactose death

A class of galactose-resistant mutants has been derived from strains of Salmonella typhimurium which are defective in uridine diphosphoglucose-4-epimerase. Resistant strains are phenotypically similar to parent organisms but do not lyse in the presence of galactose. Low levels of functional epimerase can be detected in induced cells grown at 20 C but not at 37 C, and acid is not produced from galactose. Sufficient galactose is synthesized at reduced temperatures to fabricate smooth lipopolysaccharide and acceptor sites for phage P22 from galactose-deficient media. The leaky nature of these mutants may account for resistance to galactose death by maintaining galactose metabolites at a subcritical level. Glucose protects sensitive strains by control of levels of toxic metabolites by catabolite repression.     

A Salmonella Phage-P22 Mutant Defective in Abortive Transduction

In the course of a lytic infection the Salmonella phage P22 occasionally encapsulates bacterial DNA instead of phage DNA. Thus, phage lysates include two classes of viral particles. Phage particles carrying bacterial DNA are referred to as transducing particles and deliver this DNA to a host as efficiently as particles carrying phage DNA. Once injected, the transduced DNA can either recombine with the recipient chromosome to form a ``complete'''' transductant, or it can establish itself as an expressible, nonreplicating genetic element and form an ``abortive'''' transductant. In this work, we describe a P22-phage mutant with reduced ability to form abortive transductants. The mutation responsible for this phenotype, called tdx-1, was found as one of two mutations contributing to the high-transducing phenotype of the P22-mutant HT12/4. In addition, the tdx-1 mutation is lethal when combined with an erf-am mutation. The tdx-1 mutation has been mapped to a region of the P22 genome that encodes several injected proteins and may involve more than one mutant locus. The phenotypes of the tdx-1 mutation suggest that the Tdx protein(s) normally assist in the circularization of the P22 genome and also contribute to the formation of DNA circles thought to be required for abortive transduction.     

Specialized transduction of kanamycin resistance in a Providence strain.

Properties of a transducing system with a phage able to transduce a kanamycin-resistance marker of the T compatibility group plasmid R394 at a frequency of 2 times 10(-2)/plaque-forming unit adsorbed are described. The phage was detected in Providence strain P29 transduced to kanamycin resistance by Providence phage PL25 grown on this strain harbouring the R factor. Four P29 transductants, specially selected at the lowest multiplicities of infection of the high frequency transducing (HFT) phage, were defective lysogens. They plated PL25 with an efficiency of I and only one liberated low-titre phage spontaneously or on u.v. induction. The defect in maturation function could be corrected by introduction of a wild PL25 prophage. The transducing phage was serologically frequency was increased by the simultaneous presence of homologous non-transducing phage. Transductants did not transfer the kanamycin-resistance marker by conjugation, and produced kanamycin-sensitive segregants at a moderate rate. These segregants could be transduced to kanamycin resistance by the HFT phage. Irradiation of HFT lysates by u.v. produced an exponential fall in transduction frequency. It was concluded that the defective phage transduced by lysogenization. Kanamycin-resistant transductants could themselves be transduced by streptomycin resistance by PL25 reared on a streptomycin-resistant mutant. Lysogenic transductants produced by the HFT phage did not always liberate HFT phage on u.v. induction. Possible explantations are considered.