Virulent Mutants of Bacteriophage P22 I. Isolation and Genetic Analysis

Mutants of phage P22 which form plaques on a P22 lysogen have been isolated. These virulent mutants have been classified into three groups. (i) VirA mutants arise spontaneously in wild-type stocks and form very small turbid plaques on a P22 lysogen. The single mutation responsible for VirA virulence maps near the mnt locus, one of the immunity regions of phage P22. (ii) VirB mutants do not arise spontaneously and have been isolated only from mutagenized P22 stocks. VirB mutants form small, clear plaques on a P22 lysogen. One of the VirB mutants, virB-3, was analyzed in detail. The virB-3 mutant is comprised of two mutations: K5, which maps within the c(2) gene, and Vx, which maps in the region between the c(2) and c(3) genes. Phages carrying either the K5 or Vx mutation are not virulent in themselves but mutate to VirB virulence at a frequency of 10(-5) to 10(-6). It is concluded that K5 and Vx are mutations at specific sites which together confer the ability to undergo phage development in the presence of repressor. (iii) VirC mutants are defined by a large clear plaque morphology when plated on a P22 lysogen. VirC mutants are comprised of the determinants of both VirA and VirB virulence.     

Phage Typing Reactions on Brucella Species

The nature of the phage typing reactions on Brucella species was determined by rates of adsorption and infection, one-step growth experiments, and susceptibility to lysis from without. The highest rates of adsorption and infection were obtained on smooth B. abortus cultures, and large clear plaques were produced. One or a few phage particles per B. neotomae cell killed about one-half of the cells, but some went through an infective cycle and released mature phage that resulted in production of small clear plaques. With B. suis, more phage particles per cell were required to kill, replication did not occur, and plaques were not observed. Still greater numbers of phage particles were required to cause some inhibition of growth of B. melitensis lawns. Rough Brucella cultures and species, such as B. ovis and B. canis, were not affected by the highest concentrations of phage. B. abortus cultures of intermediate colonial morphology adsorbed phage, but only a few infected cells (after a delayed latent period) released mature phage. An infected culture or colony appeared normal until spontaneous phage mutants appeared which could penetrate the cell wall more effectively than the parent phage. The mutant phage multiplied more rapidly, and the colony changed to a sticky white form.     

Identification of a Cupin Protein Gene Responsible for Pathogenicity,Phage Susceptibility and LPS Synthesis of Acidovorax citrulli

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King’s B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.     

Mapping of the c-region of phage phi80

A set of c-mutants of the phage phi80 is isolated. These mutants fit into three genes. According to plaque morphology and frequency of lysogenization of mutants, the genes were named cI, cII and cIII as it was previously done for phage lambda. Their order, determinated by mutant phage crosses, is cIII-sus326-cI-cII-sus250. Sus326 is a mutation in the gene 15, so it is probably an analogue of the N gene of the phage lambda. Thermoinducible mutants of the phage phi80 cts11 and cts12 correspond to the mutant types cItsB and cItsA of the phage lambda and they complement each other. Thus, it is supposed that phi80 phage repressor molecules consist of few protein subunits.     

ESCHERICHIA COLI Rho Factor Is Involved in Lysis of Bacteriophage T4-Infected Cells

A Rid (Rho interaction deficient) phenotype of bacteriophage T4 mutants was defined by cold-sensitive restriction (lack of plaque formation) on rho+ hosts carrying additional polar mutations in unrelated genes, coupled to suppression (plaque formation) in otherwise isogenic strains carrying either a polarity-suppressing rho or a multicopy plasmid expressing the rho+ allele. This suggests that the restriction may be due to lower levels of Rho than what is available to T4 in the suppressing strains.--Rid394 X 4 was isolated upon hydroxylamine mutagenesis and mapped in the t gene; other t mutants (and mot, as well as dda dexA double mutants) also showed a Rid phenotype. In liquid culture in strains that restricted plaque formation Rid394 X 4 showed strong lysis inhibition (a known t- phenotype) but no prolonged phage production (another well-known t- phenotype). This implies that when Rho is limiting the t mutant shuts off phage production at the normal time. Lysis inhibition was partially relieved, and phage production prolonged to varying extents depending on growth conditions in strains that allowed plaque formation. No significant effect on early gene expression were found. Apparently, both mutant (polarity-suppressing) and wild-type Rho can function in prolonging phage production and partially relieving lysis inhibition of Rid394 X 4 when present at a sufficiently high level, and Rho may play other role(s) in T4 development than in early gene regulation.     

Molecular Characterization of a Variant of Bacillus anthracis-Specific Phage AP50 with Improved Bacteriolytic Activity

The genome sequence of a Bacillus anthracis-specific clear plaque mutant phage, AP50c, contains 31 open reading frames spanning 14,398 bp, has two mutations compared to wild-type AP50t, and has a colinear genome architecture highly similar to that of gram-positive Tectiviridae phages. Spontaneous AP50c-resistant B. anthracis mutants exhibit a mucoid colony phenotype.     

A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis subsp. lactis C2.

Phage-resistant mutants, isolated from cultures of Lactococcus lactis subsp. lactis C2 infected with phage c2, did not form plaques but bound phage normally. The mutants were sensitive to another phage, sk1, although the number of plaques was reduced approximately 56% and the plaques were four times smaller. Binding to phage sk1 was reduced about 10%. Another group of phage-resistant mutants, isolated from cultures infected with phage sk1, bound normally to both phages c2 and sk1 but did not form plaques with either phage. Carbohydrate analyses by gas chromatography of the cell walls showed no significant differences in saccharide compositions between the wild-type and phage-resistant cells. However, a difference was observed in the interactions of the phage with the cytoplasmic membranes. Membranes from the wild-type cells, but not mutant cells, inactivated phage c2. Phage sk1 was not inactivated by membrane from either strain. Treatment of wild-type membranes with proteinase K eliminated the ability of the membrane to inactivate the phage, whereas treatment with mutanolysin had no effect. On the basis of this ability to inactivate the phage, a membrane protein was partially purified by gel filtration and ion-exchange chromatography. Under nondenaturing conditions, the phage-inactivating protein has an apparent Mr of approximately 350,000. The protein has an apparent subunit size of 32 kDa, which suggests that it normally exists as a multimer with 10 to 12 subunits or in association with other membrane components. It is proposed that this protein is required for phage c2 infection.     

Thermodynamic genetics of the folding of the B1 immunoglobulin-binding domain from streptococcal protein G

A method has been developed to select proteins that are thermodynamically destabilized yet still folded and functional. The DNA encoding the B1 IgG-binding domain from Group G Streptococcus (Strp G) has been fused to gene III of bacteriophage M13. The resulting fusion protein is displayed on the surface of the phage thus enabling the phage to bind to IgG molecules. In addition, these phage exhibit a small plaque phenotype that is reversed by mutations that destabilize the Strp G domain. By selecting phage with large plaque morphology that retain their IgG-binding function, it is possible to identify mutants that are folded but destabilized compared with wild-type Strp G. Such mutants can be divided into three general categories: (1) those that disrupt packing of hydrophobic side chains in the protein interior; (2) those that destabilize secondary structure; and (3) those that alter specific hydrogen bonds involving amino acid side chains. A number of the mutants have been physically characterized by circular dichroism and nuclear magnetic resonance and have been shown to have structures similar to wild-type Strp G but stabilities that were decreased by 2–5 kcal/mol. © 1995 Wiley-Liss, Inc.     

Regulation of Bacteriophage P22 DNA synthesis and repressor levels in P22cly infections.

A rifampin-resistant mutant of Salmonella typhimurium carries an altered RNA polymerase. Wild-type (c+) phage P22 displays clear plaques and a reduced lysogenization frequency on this mutant host. The cly mutants of P22 were isolated on the basis of their ability to lysogenize such mutant hosts. Two classes of regulatory events, both of which are dependent on P22 gene c1 activity, are necessary for the establishment of lysogeny in P22. The positive events culminate in repressor synthesis; the negative events cause a retardation in phage DNA synthesis. Neither the positive nor the negative events are observed in P22c+ infections of the mutant host. Both effects are found in P22cly infections of the mutant host. Observable results of both the negative and the positive events are exaggerated in P22cly infections of wild-type hosts as compared to P22c+ infections. The cly mutation apparently increases the positive and negative regulatory events so that they are detectable in the mutant host and exaggerated in wild-type hosts. Possible mechanisms that result in the high frequency of lysogenization that characterizes the cly mutation and the nature of the cly mutation are discussed.     

Transmission of bacteriophage T4 amber mutants. I. Temperature sensitivity of gene 26 T4 phage mutant amN131 multiplication in Escherichia coli B cells]

When studying the single cycle of the multiplication of gene 26 mutant amN131 of phage T4, like in temperature shift experiments, the yield of this mutant in non-permissive host depends greatly on the temperature. The burts size of phage in Escherichia coli B is found to be 3.3 phage particles at 25 degrees C, 1.6 at 30 degrees C, 0.051 at 37 degrees C and 0.0007 at 41 degrees C. In the case of permissive host (E. coli CR-63) the burst size per cell decreases from 158 to 49 phage particles at the same temperature interval. The results of the single-burst experiments indicate, that when the incubation temperature increases, the number of E. coli B cells, in which the phage particles maturate, also decreases. It results in the dependence of the transmission coefficient value on the temperature. The transmission coefficient in the conditions favourable for the maturation of the phage is found to be 0.80. It is shown by several methods that the temperature sensitivity of the multiplication of the mutant amN131 in bacterial cells is entirely due to amber mutation in genome of the phage. Therefore the amber mutants having high temperature sensitivity when maturating in non-permissive host cells exist among ordinary amber mutants of phage T4.     

The identification of the bacteriophage HP1c1 and S2 integration sites in Haemophilus influenzae Rd by field-inversion gel electrophoresis of large DNA fragments.

The resolution of high molecular weight DNA fragments by field-inversion gel electrophoresis (FIGE) demonstrate the presence of two phage (S2 and HP1c1) integration sites (attB) in the Haemophilus influenzae Rd chromosome. In a population of wild-type cells either prophage site appears to be occupied in a single cell by one to at least three, tandemly repeated, amplified phage DNA molecules. The attL of the second bacterial attachment site present in the host SmaI fragment 7 and the leftmost part of phage S2 type B DNA of its genome organization (Piekarowicz et. al., 1986) have been sequenced. A comparison of the two bacterial att sites demonstrated that their homology is limited to the core region. A comparison of the DNA sequences of phage S2 type B and HP1c1 type C revealed a 530-bp insertion in the HP1c1 type C (not present in S2 type B) in addition to DNA variants due mostly to single-base mismatches. We postulate that phage S2 and HP1c1 genome variants (A, B, and C) evolved from a single phage origin and might stem from passage history arisen through accumulation of mutations.     

Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants.

We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria. We found that bacteriophage lambda forms extremely large plaques on the dnaA seqA double mutants. On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host. However, no significant differences in intracellular phage lambda development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size. On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis. We found that the efficiency of adsorption of bacteriophage lambda to the dnaA seqA mutant cells is decreased at 0 degrees C , but not at 30 degrees C, relative to the wild-type strain. A considerable increase in the permeability of membranes of the mutant cells for beta-galactosidase is demonstrated. The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent). We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria.     

A Mutant of E. COLI That Restricts Growth of Bacteriophage T4 at Elevated Temperatures

After nitrosoguanidine mutagenesis, a Phage Host Defective (phd) mutant of E. coli HfrH was isolated that supported the growth of T4D wild-type bacteriophage at 30°, but not at 40° or higher. Eleven independent spontaneous mutants of T4 (go mutants) were isolated that overcame the growth restriction at high temperature. All of these mutants were located within three percent recombination of a gene 39 amber mutation in the clockwise direction on the standard map. In mixed infections, the representative go mutant chosen for further study seems to be recessive to its wild-type allele. Temperature-shift experiments suggested that the mutated host function involved in phage growth is a "late" function, beginning in mid-eclipse.—Electrophoresis of phage proteins labelled early and late in infection showed that under restrictive conditions early protein synthesis was normal, but that certain late proteins were absent. However, measurements of DNA synthesis showed that under restrictive conditions the amount of phage DNA synthesized, and especially the amount of DNA sedimenting as high molecular weight replicative intermediate, was reduced. Pulse-chase experiments showed that the phage DNA made under restrictive conditions was not rapidly degraded.     

A role for lipopolysaccharide in turkey tracheal colonization by Bordetella avium as demonstrated in vivo and in vitro

We isolated two insertion mutants of Bordetella avium that exhibited a peculiar clumped-growth phenotype and found them to be attenuated in turkey tracheal colonization. The mutants contained transposon insertions in homologues of the wlbA and wlbL genes of Bordetella pertussis. The wlb genetic locus of B. pertussis has been previously described as containing 12 genes involved in lipopolysaccharide (LPS) biosynthesis. Polyacrylamide gel analysis of LPS from B. avium wlbA and wlbL insertion mutants confirmed an alteration in the LPS profile. Subsequent cloning and complementation of the wlbA and wlbL mutants in trans with a recombinant plasmid containing the homologous wlb locus from B. avium eliminated the clumped-growth phenotype and restored the LPS profile to that of wild-type B. avium. Also, a parental level of tracheal colonization was restored to both mutants by the recombinant plasmid. Interestingly, complementation of the wlbA and wlbL mutants with a recombinant plasmid containing the heterologous wlb locus from B. pertussis, B. bronchiseptica, or Bordetella parapertussis eliminated the clumped-growth phenotype and resulted in a change in the LPS profile, although not to that of wild-type B. avium. The mutants also acquired resistance to a newly identified B. avium-specific bacteriophage, Ba1. Complementation of both wlbA and wlbL mutants with the homologous wlb locus of B. avium, but not the heterologous B. pertussis locus, restored sensitivity to Ba1. Complementation of the wlbL mutant, but not the wlbA mutant, with the heterologous wlb locus of Bordetella bronchiseptica or B. parapertussis restored partial sensitivity to Ba1. Comparisons of the LPS profile and phage sensitivity of the mutants upon complementation by wlb loci from the heterologous species and by B. avium suggested that phage sensitivity required the presence of O-antigen. At the mechanistic level, both mutants showed a dramatic decrease in serum resistance and a decrease in binding to turkey tracheal rings in vitro. In the case of serum resistance, complementation of both mutants with the homologous wlb locus of B. avium restored serum resistance to wild-type levels. However, in the case of epithelial cell binding, only complementation of the wlbA mutant completely restored binding to wild-type levels (binding was only partially restored in the wlbL mutant). This is the first characterization of LPS mutants of B. avium at the genetic level and the first report of virulence changes by both in vivo and in vitro measurements.     

A simple technique for the isolation of deletion mutants of phage lambda.

We describe a simple technique for isolating deletion mutants of phage lambda and use it to dissect a cloned fragment of foreign DNA. The technique is based on our previous finding that the normally essential product of lambda head gene D is dispensible for phage growth if the DNA content of the phage is less than 82% that of lambda wild-type (Sternberg and Weisberg, 1977). A significant fraction of the few phage that form plaques when a D amber mutant is plated on a nonsuppressing host contains deletions that reduce the phage chromosome size to less than 82% that of wild-type. It is possible to isolate deletions ranging in size from less than 1.5 kb to 14 kb (3 to 27% of wild-type lambda), and the size range can be restricted by an appropriate choice of the DNA content of the starting phage. This method, unlike the older EDTA or heat resistance methods, permits the scoring of deletions because of the absence of phenotypic variants. We investigated the effect of several host and phage mutations on deletion frequency and type and have determined that a host polA mutation increases the frequency of deletions about 30-50-fold without changing the type of deletions. A host mutD mutation or thymine deprivation increases deletion frequency about 10-fold. In contrast, a host ligts mutation has no effect on the frequency of deletions. We have also determined that the size of the smallest lambda chromosome packageable in a plaque-forming phage particle is 72-73% that of lambda wild-type.     

Genetic and biochemical analysis of mutation(s) affecting ricin internalization in Chinese hamster ovary cells

We have previously reported the isolation and characterization of Chinese hamster ovary (CHO) cell mutants defective in the internalization of ricin (Ray, B., and Wu, H.C. (1982) Mol. Cell. Biol. 2, 535-544). These mutants also do not exhibit the enhancement of ricin internalization by nigericin pretreatment at a low concentration, which is observed in the wild-type CHO cells. An analysis of somatic cell hybrids between the mutant and the toxin-sensitive wild-type CHO cell line shows that all of the phenotypes associated with the toxin resistance mutation are dominant in the hybrid cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]palmitic acid-labeled cell extracts from the mutant and toxin-resistant hybrid cell lines has revealed an increased incorporation of [3H] palmitic acid into two proteins with apparent molecular weights near 30,000 in the mutant and hybrid cells as compared to that in the wild-type cell line. Our studies indicate that these two fatty acyl proteins might be related to a dominant mutation(s) which results in a decreased uptake of ricin.     

Regulation of toxinogenesis in Corynebacterium diphtheriae. I. Mutations in bacteriophage beta that alter the effects of iron on toxin production

Diphtherial toxin is produced in maximal yields by Corynebacterium diphtheriae (C7(beta tox+) only when iron is present in growth-limiting amounts. Toxin production is markedly decreased under high-iron conditions. We studied the role of the bacteriophage beta genome in this apparent regulation of toxin production by iron. Using a passive immune hemolysis assay to detect toxin antigen production in individual plaques, we identified rare phage mutants that were toxinogenic in high-iron medium. Lysogenic derivatives of C. diphtheriae C7 harboring such phage mutants were constructed. The lysogens were compared with wild-type strain C7(beta) for their ability to produce toxin in deferrated liquid medium containing varying amounts of added iron. Quantitative tests for extracellular toxin were performed by competitive-binding radioimmunoassays. We identified phenotypically distinct mutant strains that produced slightly, moderately, or greatly increased yields of toxin antigen under high-iron conditions. The toxin produced by the mutant lysogens was biologically active and immunochemically indistinguishable from wild-type toxin. Complementation experiments demonstrated that the phage mutation designated tox-201 had a cis-dominant effect on the expression of the toxin structural gene of phage beta. The characteristics of the tox-201 mutation suggest that it defines a regulatory locus of phage beta that is involved in control of toxinogenesis by iron in C. diphtheriae.     

Construction of the genetic map of the polyoma genome.

Seven early mutants, three late mutants, and one plaque morphology mutant of polyoma have been mapped by marker rescue using wild-type restriction endonuclease fragments. The early mutants map between 1.0 and 26.4 units from the Eco RI site, a region previously shown to correspond to the 3'-OH termainal half of "early" RNA (Kamen et al., 1974). The late mutants as well as the plaque morphology mutant map between 26.6 and 45.4 map units, a region previously shown to correspond to the 3'-OH terminal half of "late" RNA (Kamen et al., 1974). Analysis of the genotype of rescued virus demonstrated that the modification of the mutant DNA during marker rescue was limited to the region of the genome covered by the wild-type restriction endonuclease fragment tested.     

Stalkless mutants of Caulobacter crescentus.

A stalk, a single falgellum, several pili, and deoxyribonucleic acid (DNA) phage receptors are polar surface structures expressed at a defined time in the Caulobacter crescentus cell cycle. When mutants were isolated as DNA phage phiCbK-resistant or ribonucleic acid (RNA) phage phiCp2-resistant, as well as nonmotile, strains, 5 out of 30 such mutant isolates were found not to possess stalks, but did possess inactive flagella. These stalkless mutants were resistant simultaneously to both DNA and RNA phages and did not possess pili and DNA pendent stalkless mutants. All motile revertants simultaneously regained the capacity to form stalks and susceptibility to DNA and RNA phages. It is suggested that a single mutation pleiotropically affects stalk formation, flagella motility, and coordinate polar morphogenesis of pili and DNA phage receptors. The stalkless mutants grew at a generation time similar to that of the wild-type strain at 30 degrees C. Cell size and morphology of a stalkless mutant, C. crescentus CB13 pdr-819, were also similar to those of the wild-type strain, except for the absence of a stalk. In addition, the CB13 pdr-819 predivisional cells were partitioned into smaller and larger portions, indicating asymmetrical cell division, as in the wild-type strain. From these results, it is suggested that swarmer cells undergo transition to cells of a stalked-cell nature without stalk formation and that the cell cycle of the stalkless mutant proceeds in an ordered sequence similar to that defining the wild-type cell cycle.