Vol. 123, No. 3 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Sept. 1975, p. 1043-1054 Copyright 0 1975 American Society for Microbiology

Isolation of Specialized Transducing Bacteriophage Lambda Carrying Genes of the L-Arabinose Operon of Escherichia coli B/r JIM BOULTER' AND NANCY LEE* Department of Biological Sciences, Section of Molecular Biology and Biochemistry, Santa Barbara, California 93106

University of California,

Received for publication 16 June 1975

A heat-inducible lysis-defective phage lambda (AcI857S7) has been integrated at multiple sites within the L-arabinose region (araCOIBAD) of a strain of Escherichia coli K-12 deleted for the normal lambda attachment site (AattA). The lambda phage has become integrated with opposite orientations at two

different loci within the araB gene and with the "normal" orientation (clockwise N-RA-J) at a single site in the araC gene. The burst size, spontaneous-curing frequencies, and number of prophage harbored by each of the ara secondary-site lysogens have been determined. From these secondary-site lysogens it has been possible to generate plaque-forming ara-transducing phage (Xpara) and defective ara-transducing phage (Adara), as well as defective leucine-transducing particles (Adleu). The construction and characterization of these Aara-transducing phage and their derivatives which carry genetically defined portions of the L-arabinose region are presented.

Until recently, bacterial genes carried by the specialized transducing phage lambda (A) were limited to those genes immediately adjacent to the normal prophage attachment site (attB.B') on the bacterial chromosome. The number of different bacterial genes carried by a lambda transducing phage, then, was severely limited. The discovery by Shimada et al. (17) that the bacteriophage lambda could successfully lysogenize strains of Escherichia coli K-12 deleted for the normal lambda attachment site (AattA) has provided molecular biologists with a tool for biochemical and genetic research. Using such XattA strains it has been possible to isolate stable secondary-site lysogens and specialized transducing particles which carry various segments of the bacterial chromosome (3, 9, 12). In this paper we wish to report on the isolation and characterization of specialized transducing X phages which carry genetically defined portions of the L-arabinose region of E. coli B/r strain UP1000. By directed integration of a heat-inducible, lysis-defective bacteriophage lambda (AcI857S7) into the L-arabinose region, we have been able to isolate plaqueforming ara-transducing phages (Apara), defective ara-transducing phages (Adara), and defective leucine-transducing phages (Adleu).

MATERIALS AND METHODS Bacteria and phage strains. The source and relevant genotype of each bacterial strain used in this study are presented in Tables 1 and 2. The bacteriophage XcI857S7 was obtained from P. Cleary, and XcI9Oc17 was from J. Schrenk. Asus mutants were from A. Campbell. Media. Tryptone yeast extract medium (TYE) contains 15 g of yeast extract, 10 g of tryptone, and 5 g of sodium chloride per liter. Tryptone plating agar, tryptone bottom agar, and tryptone soft agar contain the same ingredients plus 15 g, 10 g, and 6 g of agar, respectively, per liter. Yeast extract tryptone broth and yeast extract tryptone agar are similar to TYE and tryptone bottom agar except that the concentrations of yeast extract is 5 g per liter and that of tryptone is 8 g per liter. Eosin-methylene-blue agar (EMBO) is Difco formula, without addition of carbohydrate. EMBA is EMBO plus 1% L-arabinose, and EMBSA is EMBA with 6 g of agar per liter instead of 15 g. Mineral L-arabinose agar and mineral L-arabinose soft agar (MASA) have been described elsewhere (16). Tryptone-ribitol-2,3,5-triphenyltetrazolium chloride agar (TPTC-ribitol) has been described by Katz (9). All mineral media are supplemented with thiamine at a final concentration of 10 jsg per ml. Non-nutritive bacterial and phage dilution buffer

was 0.01 M tris(hydroxymethyl)aminoethane (Tris)glycine, 0.01 M MgSO4, 0.01% gelatin, pH 7.4 (17). Polyethylene glycol resuspension buffer contained 1% K,HIP0,-KH.P04 (pH 7.0), 0.02% MgSO4-7H,O, and ' Present address: Department of Biochemistry, Dart- 0.1% (NH)2SO4. Standard saline citrate buffer conmouth Medical School, Hanover, N.H. 03755. tained 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0. 1043

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BOULTER AND LEE

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TABLE 1. Nonlysogenic E. coli K-12 strainsa Designation

NL 20-000 NL 20-011 NL 20-027 NL 20-028 NL20-029 NL20-030 NL 20-036

NL 20-037 NL 20-039 NL 20-040 NL 20-041 NL 20-042 NL 20-043 NL 20-044 NL 20-045 NL 20-046 NL 20-056 NL 20-057 NL 20-058 NL 20-059 NL 20-060 NL 20-061 NL 20-071 NL 20-072 NL 20-077 NL20-078 NL 20-079 NL 20-080 NL 20-081 NL 20-082 NL 20-083 NL 20-084 NL 20-085

Relevant genotype

Source

leu lacA514Xatt+ thr araA 719 suj1 araA715 araA 766 araClOl araC12 A(gal attA chiA bioA uvrB thi A(leu-ara) AJ_P A(gal attX chiA bioA uvrB) thi araCc67 araD139 araB27 araB24 araB14 araB43 araBA87 araC3 araCl9

This study This study B. Gielow B. Gielow B. Gielow B. Gielow This study

araA2 araD53

araD139 araAl3 areA35 areA39 araA54 araA4 araA45 araB8 araB29 araBl araB25 araB46 araC98 araC97 araC124 araC75

The cultures were lysed with 0.05 ml of chloroform and centrifuged at 6,000 x g for 10 min to remove bacterial debris. All phage lysates were stored over chloroform at 4 C. Plaque-forming titers were determined on tryptone bottom agar and tryptone soft agar with the appropriate bacteria. Plaque-forming ara-transducing phages were plated on ara- recipients

TABLE 2. Lysogenic E. coli K-12 strains Designation

NL 20-800 This study

NL 20-801 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study

NL 20-802 NL 20-804 NL 20-806

This study

This study This study This study

NL 20-808

NL 20-046 (lysogenic for This study XparaBAD) NL 20-037 (polylysogenic This study for XcI857S7 integrated in araB) NL 20-028 (lysogenic for This study

XdaraCc67Bleu) NL 20-810 NL 20-811

NL 20-812 NL 20-813 NL 20-814

NL 20-817

tained 0.4% NaCl, 0.005% gelatin, 0.5 mM MgSO4, and 0.05 M Tris-glycine, pH 7.4. Preparation of phage lysates and phage DNA. Stock lysates of AcI857S7, )b2c, and Avir were prepared by the pour plate method using tryptone broth agar and tryptone soft agars and strain NL 20-011 as a permissive host. Low-frequency and highfrequency transducing lysates (LFTs and HFTs) of lambda lysogens were prepared in the following manner. Two milliliters of TYE cultures of the lysogens were grown at 32 C for approximately 18 h. Part (0.6 ml) of the culture was diluted into 4 ml of fresh TYE and grown, with shaking, for an additional 2 h at 32 C. The cultures were then shifted to 42 C for 20 to 25 min and finally incubated for 3.5 h at 37 C.

R. Weisberg

NL 20-000 (lysogenic for This study XcI857S7,

Zubay's buffer contained 0.1 M NaCl and 0.1 M con-

A(gal attX chiA bioA uvrB) thi leu (XcI857S7 integrated in leucine) NL 20-037 (polylysogenic for XcI857S7 integrated in araB) NL 20-037 (polylysogenic for AcI857S7 integrated in araB) NL 20-037 (single AcI857S7 integrated in araC) NL 20-028 (lysogenic for

NL 20-807

NL 20-815

Na2HPO4-NaH2PO,, pH 7.1. Weigle's buffer

Source

XparaCc67B)

strains, except NL 20-036, possess an ara-leu region isogenic with E. coli B/r strain UP 1000. Unless otherwise noted, all strains are Xatt+ and A sensitive. a All

Relevant genotype

NL 20-816

NL 20-820 NL 20-821

NL 20-822 NL 20-834

NL 20-835 NL 20-038 NL 20-507

XcI857S7, XdaraCc67B26) NL 20-000 (single This study Xc1857S7 lysogen) NL 20-000 (Xvirr deriva- This study tive of NL 20-812 NL 20-042 (lysogenic for This study XparaBA1) NL 20-042 (lysogenic for This study

XparaBA2) NL 20-042 (lysogenic for This study XparaBA3) NL 20-042 (lysogenic for This study AparaBA4) NL 20-028 (lysogenic for This study XparaCRB) NL 20-028 (lysogenic for This study AparaCA766B) NL 20-044 (lysogenic for This study XparaC3B) NL 20-801 (single This study XcI857S7 in araB) NL 20-810 (single This study XcI857S7 in araB) ton su1jj J. Schrenk N418 (lysogenic for P2) NL 20-011 (lysogenic for Ref. 23

A(gal-bio) mel

480AImmcI857S-)

VOL. 123, 1975

Aara-TRANSDUCING BACTERIOPHAGE

using EMBA and EMBSA, where ara+ transductants appear as dark centers within individual plaques on a pink lawn after 36 h of incubation at 32 C. The following procedure was used for all experiments requiring large quantities of A-helper (AcI857S7) or Apara transducing phage. A 500-ml TYE culture of NL 20-028 was grown overnight in a 1-liter Kluyver flask at 37 C. In the morning the absorbance at 590 nm was determined, and the cells were centrifuged at 5,680 x g for 20 min and suspended in an equal volume of polyethylene glycolresuspension buffer. The cells were infected with the desired phage at a multiplicity of 0.1 plaque-forming phage per cell and incubated at 37 C with slow shaking for 15 min. Aliquots of 100 to 200 ml of the infected cells were diluted with 9 volumes of prewarmed TYE and grown in Kluyver flasks with aeration for 3 h. At the end of this time the culture was treated of 0.01 volume of chloroform and centrifuged at 7,410 x g for 30 min. The supernatant was adjusted to contain 0.5 M NaCl and 10% (wt/vol) polyethylene glycol (average molecular weight 6,000 to 7,500). After stirring for 30 min at 4 C the phage was collected by centrifugation at 6,000 x g for 30 min. The pellets containing phage were suspended in 1/25th original culture volume of 0.01 M Tris-glycine, 0.01 M MgSO4, pH 7.5, by stirring slowly overnight at 4 C. The phage particles were purified by centrifugation in CsCl block gradients followed by isopycnic centrifugation in a CsCl solution of density p = 1.49388 at 23,000 rpm in a Spinco type 40 rotor for 20 h at 20 C. All Aara-transducing phages, with the exception of AdaraCB26, were of greater density than the parental phage. k80iA hybrid helper and 48OiA aratransducing phages were purified as described in (23). After equilibrium centrifugation the phage bands were withdrawn by puncturing the side of the tube with a hypodermic syringe fitted with a 24-gauge needle. The preparations so obtained were dialyzed against 0.01 M Tris-glycine, 0.01 M MgSO4, pH 7.4 (for the A phage preparations), or 0.1 M NaCl, 0.1 M Na,HPO,-NaH,PO4, pH 7.1 (for the 48OiA phage preparations), and stored at 4 C. Phage deoxyribonucleic acid (DNA) was extracted from purified phage as previously described (23). The separated strands of the AcI857S7 and Aara phage DNAs were obtained as in (20). Ribonucleic acid (RNA) pulse labeling and 'Hlabeled RNA-DNA hybridization assays. 3Hlabeled RNA was obtained by treating a 10-ml sample of the appropriate culture with 200 MCi of ['H Juridine (20-30 Ci per mmol) for 1 min. The cells were killed by pouring the culture into an equal volume of crushed frozen killing medium (0.02 M Tris-glycine, pH 7.3, 0.005 M MgCl,, 0.02 M NaN,, and 400 sg of chloramphenicol per ml) containing 10% (wt/vol) sucrose. The extraction and isolation of the 'Hlabeled RNA (pulse labeled) was performed as described by Rose et al. (14). The following procedure was used to isolate A 'H-labeled RNA. One milliliter of a TYE broth culture of strain NL20-812 (lysogenic for AcI857S7) was used to inoculate 200 ml of fresh TYE media and grown in a Kluyver flask at 32 C overnight. In the

morning the cells were diluted to a culture density of 1.65 x 10' cells per ml with fresh TYE media to a total volume of 100 ml. The culture was grown at 32 C until the cell density had reached 6.6 x 10' cells per ml; at this time the culture was shifted to a water bath adjusted to 42 C. Twenty minutes later, the culture was shifted to 37 C, and 10 ml of the culture were taken into 125-ml Erlenmeyer flasks containing 200 MCi of ['H]uridine and incubated for 1 min. At the end of the labeling period the cells were killed and the 'H-labeled RNA was extracted as described above. Specific RNA species were measured using liquid RNA-DNA hybridization at 65 C for 4 h as previously described (11), using separated- DNA strands of AcI857S7 and various Aara-transducing phages. Construction of t^arabinose nonutilizing derivatives of E. coli K-12. All ara- E. coli K-12 strains used in this study were constructed by growing bacteriophage Plbt on the appropriate E. coli B/r araileu+ strains and transducing the E. coli K-12 ara+leu- strain NL20-000 to leu+ on mineral glucose agar plates. The ara- markers were co-transduced with Ieu+ at a frequency of about 50% (8). The K-12 ara- derivatives were verified by crossing with appropriate F'ara's, and lysogenic transductants were eliminated after testing for restriction of Ab2c. Construction of E. coli K-12 araCc67Dl39AattA. Strain NL20-800 is an ara+ secondary-site lysogen with a temperature-sensitive lambda prophage integrated in the leucine operon (Fig. la). This strain was grown overnight in TYE broth at 32 C; in the morning the cells were diluted and plated to prewarmed EMBA plates at 37 C. Surviving ara- clones were purified on homologous media, and cross-streaked with Ab2c and Avir to identify nonlysogenic, lambdasensitive survivors. These ara-leu- strains resulted from improper excision of the temperature-sensitive prophage with a concomitant deletion of a block of genes from a point to the right of the lambda immunity region leftward into the L-arabinose operon (see Fig. lb). Each of the araleu-, A-sensitive isolates was further characterized to establish the amount, if any, of the lambda genome remaining between ara and leu as well as the extent of the leftward deletion into the L-arabinose region. The end point of the deletion in the ara region was determined by mating the isolates with F'ara homogenotes, and the residual phage genes were determined by marker rescue experiments (17) with Asus mutants. An araileu-, A-sensitive strain (NL 20-036) was selected and characterized as containing the residual phage genes AJ rightward to AattP and a deletion extending leftward into and terminating within the araBAD operon (see Fig. lb). This strain was used as a recipient in phage Plbt transduction to introduce the ara region of E. coli B/r strain and at the same time introduce an ara genotype that would assist in the effort to integrate a AcI857S7 phage in the ara genes. Phage Plbt was grown on a 1eu+araCc67Dl39 derivative of E. coli B/r and used to transduce NL 20-036 (araileu-AattA) to Ieu+ on mineral glucose plates. The Ieu+ transductants were scored for their inability to grow on mineral L-arabinose plates, due to

1045

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BOULTER AND LEE

J. BACTMOL.

(a) NL 20-800 D

~I

hr

ABC

I

II I-

RA J b2

N

I. l

offA

Ie

HEAT-INDUCTION (b) NL 20-036 D I A X

I

a

J~

~

~

b~

~

~

~

~

~

~

~

~~

Iu

aI

t

TRANSDUCTION WITH Plbt BkoraC 67 D139 IOu*

(C)

NL 20-037 D I ,

Im IaI

A

I I

I

I

I

I

C

I

{

Ott

c7I

FiG. 1. A flow-diagram showing the construction of E. coli K-12/E. coli B hybrid araCc67Dl39XattA (NL 20-037). The K-12 bacterial chromosome is represented by a straight line, the Bir bacterial chromosome by a heavy line, and the prophage DNA by a wavy line. Strain NL 20-800 harbors the XcI857prophage in the leucine operon; the prophage gene order was taken from Shimada et al. (17). Heat induction of this strain resulted in the isolation of NL 20-036, which is missing the right portion of the ara region and most of the prophage genes. This strain was crossed with a Plbt lysate of E. coli B/r araCc67Dl39 to introduce the E. coli B/r ara region, yielding the strain NL 20-037.

the presence of the araDl39 mutation, and, by assay- for ara+ transductants after 48 to 72 h of incubation at ing uninduced cells for high levels of L-arabinose 32C. Determination of the properties of the ara seisomerase activity, for the presence of the constitutive araCc67 allele (see Fig. lc). One transductant, NL ondary-site lysogens. The burst size and spon20-037 (araCc67DI39XattA), was used in subsequent taneous-curing frequencies for the secondary-site lysogens were determined using A-vir resistant dework. Isolation of secondary-site lysogess with the rivatives of the lysogens as detailed by Shimada prophage integrated in the L-arabinose genes. The et al. (17). The residual phage genes in a prophage presence of the araCc67 allele in the XattA strain deletion mutant were determined as described by provides a means for direct selection for prophage Shimada et al. (17), using approximately 10' plaqueinsertion in the ara genes, since the constitutively forming units of various Xsus mutants. To determine produced L-ribulokinase (araB) renders the strain the number of XcJ857S7 prophages in each of the sensitive to the presence of ribitol in the growth ara secondary-site lysogens, log-phase cultures of NL medium (9). The ribitol inhibition in strain NL 20-037 20-801, -802, -804, and -810 were cross-streaked on can be relieved if either the araB (L-ribulokinase) or EMBO agar media with high-titer lysates (10' to the araC (regulator) gene were inactivated by the 101' plaque-forming phage per ml) of the virulent mutant bacteriophage Xc9Ocl7. The plates were inintegration of a AcI857S7 phage. NL 20-037 was grown ovemight in TYE broth to a cubated at 32 C for 15 to 18 h and scored for culture density of 2 x 10' cells per ml. The cells were lysis. Appropriate single (NL 20-836) and double harvested by centrifugation and suspended in an (NL 20-837) XcI857S7 lysogens were used as controls equal volume of buffer (0.10 M Tris-glycine, 0.10 M on each plate. Single lysogens are sensitive to the MgSO4, and 0.10% gelatin, pH 7.4). The cells were WcO9c17 phage, while multiple lysogens are resistant mixed with XcI857S7 at a multiplicity of infection of 2 (18). to 5 plaque-forming units per cell. After 20 min of Heat-pulse curing of the polylysogenic ara secadsorption at 32 C, aliquots of the mixture were ondary-site lysogens. Three drops of a log-phase spread on TPTC-ribitol plates and incubated at 32 C culture of NL 20-801 and NL 20-810 were placed into for 18 to 36 h. In some cases the TPTC-ribitol plates fresh TYE media (5 ml) and incubated at 42 C for 6 were seeded with 2 x 10' Wb2c phage. After incuba- min. Three drops of the heat-treated cultures were tion, large red (ribitol-resistant) clones were purified diluted into 5 ml of room-temperature TYE broth and on homologous media. Each purified clone was grown incubated for 3 to 4 generations at 32 C (approxiin 1 ml of TYE broth for 6 to 8 h at 32 C, and mately 2 h). The cultures were diluted and plated to cross-streaked with Xb2c and Xvir to identify the tryptone plating agar media at 32 C for 18 h. Wellstable lysogens. These were then screened for their isolated single clones were then picked from each ability to give rise to Aara-transducing phage. Lysates tryptone plating agar plate and used to inoculate 1 ml of each of the lysogenic isolates were prepared and of TYE broth. These cultures were grown at 32 C for spotted on MASA lawns seeded with known E. coli 15 h and cross-streaked with Ac90cl7 on EMBO agar K-12 ara- mutants. The lysate spots were examined plates at 32 C. Those isolates that were sensitive to

1047

VOL. 123, 1975

Xara-TRANSDUCING BACTERIOPHAGE

lysis by Xc90c17 were considered to be single-lysogen derivatives of the parental polylysogenic ara secondary-site lysogens. Isolation of XSpi- variants. A fresh lysate was prepared from singly lysogenic strains with prophage integrated within ara, and approximately 101 to 107 plaque-forming units of each lysate were mixed with 0.1 ml of a log-phase culture of a phage P2 lysogen of E. coli K-12 (NL 20-838). After 15 min of adsorption at 37 C, 3.3 ml of melted tryptone soft agar media was mixed with the phage-infected cells, and the contents were poured onto a prewarmed tryptone plating agar plate. The plates were incubated at 37 C ovemight. In the morning, 10 well-isolated single plaques were picked with capillary tubes into 0.5 ml of TYE broth and solubilized by vortexing. To this was added 0.3 ml of a log-phase culture of the P2 lysogen and 3.3 ml of melted tryptone soft agar. The contents were poured onto a tryptone bottom agar plate and incubated at 37 C for 5 to 6 h. These plate lysates were harvested and stored over chloroform at 4 C.

TABLE 3. Characteristics of the ara-secondary-site lysogens SensiStrain

NL20-801 NL20-802 NL20-804 NL20-810 NL 20-834 NL 20-835 NL20-813d

Burst-sizea

tivity XcJ9Oto

119 140 0.001 130 0.01 1 144

r r s r s s s

Spontaneous-

curing

frequencyc 2 x 1.5 x 1 x 1 x

10-' 10-7 10-8 10-7

1.2 x 10-1

RESULTS

aDefined as the number of plaque-forming phage released per bacterium. b The sensitivity (s) or resistance (r) to the virulent AcI9Oc17 was determined as described in Materials and Methods. c Defined as the ratio of the bacterial titer at 43 C to that at 32 C. d A wild-type XcI857S7 lysogen of E. coli K-12.

Of approximately 600 ribitol-resistant clones isolated from NL 20-037, four were stable lysogens which, upon heat induction, yielded LFTs capable of transducing various arac point and deletion mutants to ara+. These isolates were designated NL 20-801, -802, -804, and -810. The observation that these strains gave rise to LFTs for ara genes and were resistant to growth inhibition by ribitol indicated that they were probably secondary-site lysogens with prophage integrated in the L-arabinose region. The experiments described below show the location and the orientation of prophage in these strains. Properties of the ara secondary-site lysogens. The spontaneous-curing frequencies (defined as the ratio of the bacterial titer at 43 C to that at 32 C) for the secondary-site lysogens were determined using Xvir-resistant derivatives of the lysogens as described by Shimada et al. (17). The values presented in Table 3, 2 x 10-7 down to less than 1 x 10', are similar to those reported for other secondary-site lysogens (10, 17, 18) and suggest that these secondarysite lysogens in ara are extremely stable. Reported burst-sizes for various secondarysite lysogens range over 6 orders of magnitude, from 2 x 102 (18) down to 1 x 10-' (10). According to Shimada et al. (17), the number of phage particles released after induction depends on the number of prophage in the lysogen, i.e., whether the secondary-site lysogen is single or multiple. No single secondary-site lysogen reported has released more than 3 phage per cell. It has been postulated that the very low burst size for the single secondary-site lysogens reflects the inefficient int and xis promoted

excision from lysogens harboring X phage at bacterial sites other than attB.B' (18). NL 20-801, -802, and -810 had burst sizes ranging from 1.2 x 102 to 1.4 x 102 (Table 3). These very high burst sizes suggest that these lysogens harbor more than one XcI857S7 phage and that the efficient phage release observed is a consequence of excision by end-cutting (24). The very low burst size for NL 20-804, less than 0.001 phage per bacterium, indicates that this secondary-site lysogen probably harbors a single prophage at the secondary site in ara. Further evidence for polylysogeny was obtained when the ara secondary-site lysogens were cross-streaked with XcI9Oc17 on EMBO plates. XcI9Oc17 is a virulent mutant bacteriophage lambda that will form plaques on single lysogens but cannot plaque on multiple lysogens (18). The results, presented in Table 3, demonstrate that NL 20-801, -802, and -810 are resistant to AcI9Oc17, and hence are polylysogenic, while NL 20-804 is sensitive to XcI9Oc17 and is, therefore, a single lysogen. It has been possible, by a brief heat treatment (see Materials and Methods), to cure both NL 20-801 and NL 20-810 of one of the prophages. These singly lysogenic derivatives, NL 20-834 and NL 20-835, respectively, are now sensitive to lysis by AcI90c17, have markedly lower burst sizes than the parental polylysogens (see Table 3), and are still able to produce, upon heat induction, LFTs capable of transducing ara genes. Mapping of the prophage. The locations of prophage in the singly lysogenic, secondary-site lysogens NL 20-804, NL 20-834, and NL 20-835 were determined by testing the bacterial and

1048

BOULTER AND LEE

J. BACTERIOL.

phage genes remaining in cells which survived incubation at high temperature (15). Each secondary-site lysogen was grown overnight in yeast extract tryptone broth medium, and the cells were concentrated by centrifugation and resuspension in 1/10th the original culture volume. The heavy cell suspension was then spread on yeast extract tryptone agar plates at 42 C. Survivors were purified, and A-sensitive clones were examined for residual phage genes by crossing with various Xsus stocks and for residual bacterial genes by crossing with appropriate F'ara homogenotes (Fig. 2). It appears that NL 20-804 contains a prophage integrated near the left end of araC, while NL 20-834 and NL 20-835 contain prophages integrated in the araB gene at two different locations. In addition, the above results suggest that the prophage in NL 20-804 and NL 20-835 are orientated clockwise while the prophage in NL 20-834 assumes a counterclockwise orientation. This is substantiated by the results obtained with their Spi- derivatives (18). Wild-type A will not plaque on a strain of E. coli lysogenic for the phage P2 (wild-type A is sensitive to P2 inhibition or Spi+). Mutants of A can be isolated at frequencies of 10-1 to 10-4 that

are

insensitive to P2 inhibition and will, therefore, plaque on a P2 lysogen (ASpi- mutants). Genetic analyses of these ASpi- mutants have shown that they are deleted for several phage genes at the left end of the prophage map, having lost as much as 15% of the A genome in the region corresponding to the xis, int, and attP sites (12). When such ASpi- mutants are isolated from lysates of a secondary-site lysogen, the deleted genes may be replaced by bacterial genes to the right of the point of prophage integration (18) (see Fig. 3). Lysates were prepared from NL 20-804 and single AcI857S7 derivatives of NL 20-801 (NL 20-834) and NL 20-810 (NL 20-835) and plated on an E. coli strain lysogenic for P2 (NL 20-838). Lambda Spi- isolates were picked, purified, and mapped as described in Materials and Methods. The results of the ASpi- transductions, (Table 4) demonstrate that from 50 to 80% of the ASpi- mutants isolated by this procedure carry bacterial ara genes and are capable of transducing E. coli K-12 ara- mutants to ara+. Given the prophage orientation and map position in each of the ara- secondarysite lysogens (Fig. 2), one would expect that the ASpi- variants isolated from NL 20-804 (XcI857S7 integrated in araC) would be capable of transducing only araC mutants, that the ASpi- mutants isolated from NL 20-835

(XcI857S7 integrated in araB) would transduce those ara - mutations to the right of araB43, and that the ASpi - mutants isolated from NL 20-834 (AcI857S7 integrated in araB) would transduce ara- mutations to the left of araB8. The data presented in Table 4 provide strong corroborative evidence that the prophage gene orders determined by deletion mapping are indeed the correct orientations for the ara secondary-site lysogens. Figure 3 illustrates (after Shimada et al. [18]) how an ara-transducing ASpi- mutant could be produced from a secondary-site lysogen in araB having the normal prophage orientation of (clockwise) N-RA-J. Isolation of plaque-forming and defective ara transducing phage (AparaBAD, AparaCc67B, and Adara Cc67B26). The effort to isolate plaque-forming ara-transducing phage was facilitated by the technique employing EMB soft agar described in Materials and Methods. This technique permitted a rapid screening of a large number of HFTs for the presence of Apara's by allowing one to score single phage for the ability to form plaques as well as to transduce ara genes. An LFT of NL 20-804 (AcI857S7 integrated in the araC gene) was used to transduce E. coli K-12 araA2 Aatt+ (NL 20-046) on mineral Larabinose agar plates at 32 C. Forty ara+ transductants were picked, purified on homologous media, and used to prepare HFTs. Each HFT was spotted on E. coli K-12 Aatt+ strains containing araB27, araB43, araBA87, araA39, araA35, and araA2. Eight of the 40 HFT examined showed a confluent growth of ara+ transTABLE 4. Transduction by ASpi- mutantsa E. coli K-12 ara recipient

No. of Parental

lysogen

NL 20-804 NL 20-834

XSpilysates

ara

0 0 + 0 0 0

0 0 + 0 0 0

araB8

ara

ara

C19

0 0 0 0 + 0

+ 0 0 0 + 0

10 (5) (5) 10 (7)

(3) NL 20-835

araA2

tested aa2B43 aa8C12

10 (8) (2)

+ 0 0 0 + 0

aThe methods used for the isolation of the ASpiphage and the procedure used in the transductions are presented in Materials and Methods. The numbers in parentheses represent the number of XSpi- lysates (out of a total of 10) giving the indicated response in transductions with the ara- recipients. The titers of the ASpi- lysates were approximately 5 x 101 plaqueforming phage per ml. +, ara+ recombinants observed under the lysate spot; 0, no ara+ transductants observed after 72 h of growth at 32 C.

ara A

ara D

ara ara I O

ara B

N

A O R A

P

101 124 19

39a87 14 24 27 I

4-a

I)(1) E

_I 4-

_

(I )C

4-4-

4.

-

ara ara A 8

ara b

-i..

D A R 0

I

P

N

4-+--+

+--

ara A I

-.- w4. 4- 4- -i..

-4-

aroara ara

10

B

C

I

14 124 27'

4-

ara D

4-

1

39 d87

(1)-

+ -0-4-4-4-

ara

J

.3D

C

Ora C

J

D I

ara B

_I

-

+--- +

I

+-4+

I-

-

-

-

-

- -

--

X

N

P

R

0

I4

D

I

J B 2427

394a87 14 .1-4-1

(1)1

.4-

4.+

4- 4-

-4-

-0- 4-

14

ara

A

101 124 19 -4 +.

rara ara IPI C 101124 19 4-

FIG. 2. Determination of prophage location and orientation by deletion mapping. Heat-cured survivors selected from the secondary-site lysogens were crossed with F'ara's containing various ara point mutations, and scored for ara+; and also crossed with lysates of various Asus mutants, and scored for lysis. The apparent prophage locations and orientations are shown for 20-804 (a), 20-834 (b), and 20-835 (c), with the results from the crosses shown below. Each deletion is represented one or more times among the heat-cured survivors, as indicated by the numbers in brackets to the left. Data obtained with deletions which do not involve neighboring bacterial genes, and those which eliminate the entire prophage, are not shown. The prophage placement within araC in 20-804 is based on the finding that this strain does not complement an F'araC to ara+, even though no known araC point mutation lies to the left of the prophage. This test does not rule out the possibility, however, of the prophage being located in the regulatory region for the araC gene (22). R

A

x THR

D

a

A

C

LEU

-~~~~~~~~~~

-if

in1 t

int

+ xis

FIG. 3. The integration of XcI857S7 into the araB gene and the formation of Xara-transducing phage. The transducing phage are of two types: XaraBAD (Agal-type transducing phage) and XaraCB (Xbio or XSpi-type transducing phage). The drawing is not to scale and is meant as an aid to material presented in the text. Abbreviations: thr, threonine; leu, leucine; D, araD gene; A, araA gene; B, araB gene; C, araC gene; A.A', the secondary attachment site in araB; P.P, the attachment site on the AcI857S7phage; int and xis, the integration and excision proteins specified by the AcI857S7 phage (after Shimada et al. [18]). Arrows represent mRNA transcripts from the phage leftward promoter poL and the bacterial promoter pBAD. The 5' to 3' transcriptional polarity dictates that both transcripts should be complementary to the same strand of DNA from the transducing phage. 1049

1050

BOULTER AND LEE

J. BACTERIOL.

ductants within the lysate spot for each of the above crosses and were tested for the presence of Xpara's as described above. All eight HFTs contained plaque-forring ara-transducing particles. One of the ara+ transductants, NL 20-808, used for the preparation of the HFTs was particularly stable (less than 0.01% arasegregants on EMB L-arabinose plates) and yielded Xpara lysates with 4 x 1010 to 6 x 1010 plaque-forming ara-transducing phages per ml. Mapping data (Table 5 and Fig. 4) demonstrate that this Xpara phage, hereafter referred to as

XparaBAD, contained very little araC, and extended leftward to include all of araB, araA, and probably araD. The following procedure was used to isolate a Xara-transducing phage which carried a functional araC gene. An LFT of NL 20-801 (XcI857S7 integrated in the araB gene) was used to transduce E. coli K-12 araA766 (NL 20-028) on mineral L-arabinose plates at 32 C. Forty ara+ transductants were picked, purified, and used to prepare HFTs. Each HFT was spotted on MASA lawns seeded with araB27, araA2,

TABLE 5. Mapping data for the Xara transducing phagea E. coli K- 12 ara- recipient strain Xara phage

D139 D53 A2 A54 B43 B14 B46 B24 B27 C124 C97 C12

XparaCc67B (NL 20-806) XparaBAD (NL 20-808) XdaraCc67B (NL 20-811) XparaC+B (NL 20-820) XparaC3B (NL 20-822) XparaCA766B (NL 20-821)

0 0 0 0 0 0

0 R 0 0 0 0

0 C 0 0 0 0

0 C 0 0 0 0

R C 0 R R R

R C 0 R R R

D139 D53 A2 A 13 A45 A35

A4

0 C 0 0 0 0

R C R R R R

R C R R R R

C 0 C

C R R

C 0 0 C C C C R R R R C

C3

C19

C

C 0 0 C C C C 0 R 0 0

A39 A54 BA87 B43 B27 C124 C97

0 XparaBA4 (NL 20-817) C 0 R R R R R R R C C R 0 XparaBA3 (NL 20-816) 0 R R R R R R C 0 0 C C R 0 0 XparaBAl (NL 20-814) 0 0 0 R R R R R C C C R 0 0 0 0 XparaBA2 (NL 20-815) C 0 R R R R 0 C C R 0 a A fresh HFT lysate was prepared for each Xara transducing phage and was spotted on MASA agar media seeded with E. coli K-12 ara mutants as detailed above. The plates were examined for ara+ transductants after 72 h of growth at 32 C. Symbols: C, confluent growth of ara+ growth under the lysate spot; R, some ara+ recombinants; and 0, no ara+ transductants. An illustration of the relative amounts of ara genes harbored by each of the Xara-transducing phage is presented in Fig. 4. ......-

n

NL 20-808 NL 20-817 NL 20-816 NL 20-814 NL 20-815

NL 20-806 m~~~~~~~~~~~~~ NL 20-821 NL 20-811 139 . 2. 13453543954 53 . 8764314462427.. 124125 19 aa . _ . 1 . I 1 = ara A ara B '' 0 aOra D . ara C[ .___ A ,,,W.* w _w ....

A

43

14

462529 z2y' ./o I .4. . rI * I I

101

i a I I2

NL 20-806 NL 20-821

i N2 NL 20-811

*

124

Nt.N.20-808

12

i

FIG. 4. The L-arabinose region (araDABIOC) of E. coli Bir illustrating the relative amounts of the ara genes carried by each of the Xara-transducing phage described in the text. The solid squares at the terminus of each "bar" represents the point of integration of the XcI857S7 phage in the araB and araC genes; the bars themselves are to illustrate the amount of ara genes carried by each Xara phage. All ara genes, except the aral and araO sites, are drawn to scale. The strain designations (e.g., NL 20-808, NL 20-821, etc.) are the numbers assigned to the host bacterium which is lysogenic for each of the various Xara-transducing phage (see Table 2 for a complete description of these strains and the Xara phage harbored by each).

VOL. 123, 1975

araA 766. Thirteen of the 40 HFTs gave a confluent growth of ara+ transductants under the lysate spot with araA766, showed numerous ara+ recombinants with araB27, and gave no ara+ recombinants with araA2. Eleven of the 13 HFTs were also plaque-forming ara-transducing lysates. One of the ara+ transductants used for the preparation of HFTs, NL 20-806, yielded Xpara lysates with 1 x 1010 to 2 x 1010 plaqueforming ara-transducing phages per ml. Mapping data (Table 5 and Fig. 4) demonstrate that this Xpara phage, hereafter referred to as XparaCc67B, contained the region defined by araB14 to araIO, and all of araC. Efforts using similar techniques to isolate Xpara phage from NL 20-810 (XcI857 integrated in araB) were unsuccessful; however, it was possible to isolate defective ara-transducing particles from this secondary-site lysogen. Mapping data (Table 5 and Fig. 4) demonstrate that this Xdara phage, hereafter referred to as XdaraCc67B26 (strain NL 20-811 is doubly lysogenic for XcI857 and XdaraCc67B26), contained the region from araB24 rightward through araIO and including all of araC. We propose that these ara-transducing phages have been generated by improper excisions in the same manner as in the case of other transducing phages (2). The recombination events resulting in the circularization of phage DNA are shown in Fig. 3. According to the scheme depicted, a Xara-transducing phage derived from a prophage integrated clockwise within the ara region would carry the araBAD operon in the same orientation as the phage promoter XpoL. This would be the case in XparaBAD and XdaraCB26. On the other hand, a Xara-transducing phage derived from a prophage integrated counterclockwise in the ara region would carry the araBAD operon with the transcriptional polarity of the phage promoter XpoR. One would, therefore, expect a messenger of RNA (mRNA) transcript of the araBAD operon to hybridize preferentially to the I-strand DNA of XparaBAD and XdaraCB26, and to the r-strand DNA of XparaCB. This is tested as described below. The DNA of ara-transducing phages is readily separated into "H" and "L" strands upon denaturation in the presence of the polyribotide poly(U,G) (Fig. 5). 9H-labeled RNA obtained from the hyperinducible strain F'araB24/B24 hybridizes preferentially to the L strands of XparaBAD and XdaraCB26, and to the H strand of XparaCB (Fig. 6). To ascertain whether these operationally defined H and L strands do indeed correspond respectively to the "r" and "I"

Xara-TRANSDUCING BACTERIOPHAGE

1051

0

(D N

FRACTION NUMBER FIG 5. Separation of the heavy (H) and light (L) strands of the Xara-transducing phage DNA by centrifugation to equilibrium in cesium chloride gradients containing the RNA copolymer U,G. (a) Separation of the H and L strands of XparaBAD DNA (NL 20-808), (b) XdaraCc67B26 DNA (NL 20-811), and (c) AparaCc67B DNA (NL 20-806). Bars ( indicate the fractions pooled. In each case the direction of sedimentation is from right to left. Sample preparation and the conditions for sedimentation are detailed in Materials and Methods.

strands as is known for lambda, 9H-labeled RNA from a heat-induced lysogen of lambda XcI857S7 was obtained as described in Materials and Methods, and labeled transcripts of r and I strands were isolated by selective hybridization to separated strands of XDNA (1). These r and l transcripts were recovered and used to test the H and L strands of ara-transducing phages. Results shown in Fig. 7 clearly indicate that the H and* L strands of the transducing phages correspond to the H and L strands of the parent phage, and the addition of bacterial DNA with or without the loss of a portion of the phage genome in these instances did not result in a reversal of the bouyant density of r and I strands of DNA under these conditions. It thus appears that the araBAD operon is orientated to read leftward in XparaBAD and XdaraCB26, and rightward in XparaCB. Isolation of defective leucine-transducing

1052

BOULTER AND LEE

J. BACTERIOL.

phage Adleu. An LFT of NL 20-801 (XcI857S7 integrated in the araB gene) was used to transduce E. coli K-12 araA766 (NL 20-028) on mineral L-arabinose plates at 32 C. HFTs prepared from 40 ara+ transductants were spotted on MASA lawns seeded with E. coli K-12 ara+leu- (NL 20-000) to determine if any of the ara-transducing phages carried, in addition to the ara genes, bacterial genes responsible for the biosynthesis of leucine. Three of the 40 HF'Ts tested showed a confluent growth of leu+ transductants when spotted on MASA lawns seeded with NL 20-000. All three leu-transducing A phages are defective showing a requirement for AcI857S7 helper-phage to obtain Ieu+ transduction. Construction of additional Aara phage. We wish to report on two selection procedures that have been used in (i) the construction of derivatives of AparaCc67B that carried previously characterized mutations in the araC gene and (ii) the construction of derivatives of AparaBAD which contain lesser amounts of araBAD operon DNA. When an araC- cell is lysogenized by AparaCc67B, the lysogen is ara+ and ribitol-sensitive. Selection on ribitol-TPTC plates allows E

1.5

'b)

H

E

cs

0 v

N

m I N

0

DNA , ug

E

CL 0

w

a N

'a I Nx

C0 DNA , ug

Cc)

L L6

0~~~~~~~~~~~~~~~ w 13.2 I

E

,,

I~~~~~~~~~~~~~~~

c

FIG. 6.Tesrn.pcfciyo3nvv

yte

.0

ztion

1

12 8

L

0

/ CB26L

16

w

N

4

CB26L

0 3

0

1

30

1

2

3

f HlbdaamN ofcntn2mut

0

to Hnrasn DNA fAr-rndcn hg 2mut ug

2

0

3

,

The strand

specificity of in vivo syntheas measured by hybridization of constant amounts of 3H-labeled ara-mRNA to increasing amounts of Xara-transducing phage FIG.

sized

6.

DNA

3H-labeled

ara-mRNA

DNAs. Pulse-labeled 3H-labeled ara-mRNA isolated from F'araB24/araB24 was hybridized to increasing amounts of (a) AparaCc67B H- and L-strand DNA, (b) XdaraCc67B26 H- and L-strand DNA, and (c) AparaBAD H- and L-strand DNA. The number of counts per minute hybridized to the various Aara Hand L-DNA strands in excess of XcI857S7 (wild type) H- and L-strand DNA is considered to be ara mRNA. The number of counts per minute hybridized to each of the Xara H- and L-strand DNAs should not be taken as an indication of the relative amount of ara DNA carried by each of the phage as the results presented are taken from experiments utilizing 3Hlabeled RNA with different specific activities.

,

ug

FIG. 7. The strand specificity of pulse-labeled 'Hlabeled A-RNA as measured by hybridization assays with constant amounts of 'H-labeled AH-RNA (solid circles) and 'H-labeled AL-RNA (open circles) with increasing amounts of (a, d) AparaBAD H- and L-strand DNA, (b, e) XparaCe67B H- and L-strand DNA, (g) XdaraCc67B26 L-strand DNA, and (c, f) wild-type AcI857S7 H- and L-strand DNA. Each hybridization assay contained 50 Al of 'H-labeled AH-RNA (10,500 counts/min) or 'H-labeled AL-RNA (9,890 counts/min) in a total volume of 400 A/l. to isolate lysogenic derivatives carrying the chromosomal araC- allele. In this manner, AparaC3amwr (in NL 20-822) and AparaCA766 (in NL 20-821) were constructed. We have also

one

VOL. 123, 1975

isolated what appears to be a partial revertant XparaC+ B (in NL 20-820) in this manner. This strain is ribitol resistant, ara+, and has regained the D-fucose sensitivity of the wild-type araC+ allele (5). It is probably not a true reversion to C+, since its ability to program the synthesis of a functional araC product in an in vitro DNA-dependent protein synthesizing system is only YMoth that of a template carrying a wildtype araC gene (22). Derivatives of the XparaBAD phage (NL 20-808) which harbor lesser amounts of araBAD operon DNA were isolated as follows. A fresh HFT of NL-20-808 was used to transduce E. coli K-12 araB43 (NL 20-042; Fig. 4) on mineral L-arabinose agar plates. Ara+ transductants were purified and used to prepare HFTs. Each of the HFTs was spotted on MASA lawns seeded with ara- point mutants as described in Table 4. Of several hundred HFTs examined a small number of HFTs (about 5%) were able to complement all araB point mutants (and hence carried all of araB plus araI) but would recombine only with some araA point mutants. The rest were identical to parental phage. Presumably, during excision of the XparaBAD prophage occasional Xara-transducing phages have been generated which contain less araBAD operon DNA than the parental XparaBAD phage. It is not clear, however, why these Xara phage are generated at such a high frequency. It is possible that the XparaBAD phage carries a large amount of bacterial DNA (outside of the ara region, between threonine and araD [21 ]) that is highly susceptible to nucleolytic cleavage during, or prior to, the packaging of the lambda genome by the coat protein. In any event, it has been possible by this technique to delete from araD to parts of araA (from left to right) portions of the araBAD operon DNA from the XparaBAD phage. These plaque-forming aratransducing phage, carrying part of araD, all or part of araA, and all of araBIO, are designated XparaBA phage. Figure 4 illustrates the relative amounts of gene araD and araA that each of these isolates possesses. DISCUSSION The directed integration of XcI857S7 at multiple sites within the L-arabinose operon of E. coli B/r is reported. The prophage has been integrated with opposing polarities at two different sites in the araB gene and at a single site with the normal orientation in the araC gene. Various lambda transducing particles have been derived from these secondary-site lysogens that are proving to be extremely useful in studies on

Xara-TRANSDUCING BACTERIOPHAGE

1053

the expression of the L-arabinose operon. Already selected Xpara phage have made it possible to determine the polarity of transcription of the araC gene (the gene coding for the positive control protein of the L-arabinose operon) (22); have assisted in the isolation, purification, and sequencing of short segments of ara mRNA (unpublished data); have helped define the mechanism of araC activation of the Larabinose operon (11); and have been useful in exploring the kinetics of ara-specific mRNA synthesis under various physiological conditions (unpublished data). Derivatives of these phage harboring deletions of the ara-controlling sites (araIO deletions) are being constructed in an effort to determine, by heteroduplex analyses, the physical site of this region. It now appears possible that, with appropriate selection techniques, lambda may be integrated at any one of a large number of secondary sites on the E. coli chromosome (10, 17). Shimada et al. (17) have performed a comprehensive analysis of several thousand secondary-site lysogens and the results of their work strongly suggest that the integration of X at the various attA.A' sites is not a completely random event and that there exists an order of preference among secondary sites for X lysogenization. Our results indicate that the three discrete secondary sites located within ara do not differ significantly in their affinities for A. While the physical identity of these sites cannot be assessed from the available information, we noted that if single lysogen derivatives of the ara secondarysite lysogens are cured of the prophage, it is possible to regenerate the original ribitol-sensitive phenotype of NL 20-037. This is consistent with a mechanism of int-promoted recombination in which the nucleotide sequence of the secondary sites (attA.A') within araC and araB are restored after a cycle of insertion and excision (6). The selection procedure employed in this study required that the phage integrate in a segment of DNA (the araB and araC genes) consisting of approximately 2,100 nucleotide base pairs (not including the araIO region located between araB and araC). A calculation of the number of times a specific sequence of nucleotide base pairs would occur in a 2,100 base pair stretch of DNA shows that a four-base sequence would be expected to repeat itself 8.3 times, and a five-base sequence would be expected to repeat 2.2 times, whereas a six-base sequence would be expected to repeat itself only 0.5 times. These calculations suggest that the longest specific sequence of bases that might be

1054

BOULTER AND LEE

expected to repeat itself three times (the number of attA.A' sites found in the araB and araC genes) in 2,100 base pairs is five. Based on analyses of attB.P' and attP.P' heteroduplexes, Davis and Parkinson (4) have suggested that the normal attB.B' locus may be less than 20 base pairs in length. It is of interest to note that very recently Shimata et al. (19) have reported a calculated length of five or six base pairs for the size of the secondary attachment sites for

J. BACTERIOL.

11.

12.

13.

x.

14.

ACKNOWLEDGMENTS This investigation was supported by U.S. Public Health Service grant GM-14652 from the National Institute of General Medical Sciences. We wish to thank R. Iwata for technical assistance.

1.

2. 3.

4.

5. 6.

7.

8.

9. 10.

LITERATURE CITED B0vre, K., and W. Szybalski. 1971. Multistep DNA-RNA hybridization techniques, p. 350-383. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 21. Academic Press Inc., New York. Campbell, A. 1962. The episomes. Adv. Genet. 11:101-145. Chung, S. T., and R. Greenberg. 1973. Loss of an essential function of Escherichia coli by deletions in the thyA region. J. Bacteriol. 116:1145-1149. Davis, R., and J. Parkinson. 1971. Deletion mutants of phage lambda. III. Physical structure of atto. J. Mol. Biol. 56:403-423. Englesberg, E., and G. Wilcox. 1974. Regulation: positive control. Annu. Rev. Genet. 8:219-242. Franklin, N., W. Dove, and C. Yanofsky. 1965. The linear insertion of a prophage into the chromosome of E. coli by deletion mapping. Biochem. Biophys. Res. Commun. 18:910-923. Gottesman, M. E., and R. A. Weisberg. 1971. Prophage insertion and excision, p. 113-138. In A. D. Hershey (ed.), The bacteriophage lambda, Cold Spring Harbor Press, New York. Gross, J., and E. Englesberg. 1959. Determination of the order of mutational sites governing L-arabinose utilization in Escherichia coli B/r by transduction with phage Plbt. Virology 9:314-331. Katz, L. 1970. Selection of araB and araC mutants of Escherichia coli BIr by resistance to ribitol. J. Bacteriol. 102:593-595. Kirschbaum, J. B., and B. Konrad. 1973. Isolation of a

15. 16.

17.

18.

19.

20.

21. 22. 23.

24.

specialized lambda transducing bacteriophage carrying the beta-subunit for Escherichia coli ribonucleic acid polymerase. J. Bacteriol. 116:517-526. Lee, N., G. Wilcox, W. Gielow, J. Arnold, P. Cleary, and E. Englesberg. 1973. In vitro activation of the transcription of araBAD operon by araC activator. Proc. Natl. Acad. Sci. U.S.A. 71:634-638. Lindahl, G., G. Sironi, H. Bialy, and R. Calendar. 1970. Bacteriophage lambda; abortive infection of bacteria lysogenic for phage P2. Proc. Natl. Acad. Sci. U.S.A. 66:587-594. Lis, J. T., and R. Schlief. 1973. Different cyclic AMP requirements for induction of arabinose and lactose operons of Escherichia coli. J. Mol. Biol. 79:149-162. Rose, J., R. Mosteller, and C. Yanofsky. 1970. Tryptophan messenger ribonucleic acid elongation rates and steady-state levels of tryptophan operon enzymes under various growth conditions. J. Mol. Biol. 51:541-550. Shapiro, J., and S. Adhya. 1969. The galactose operon of E. coli K-12. H. A deletion analysis of operon structure and polarity. Genetics 62:249-264. Sheppard, D. E., and E. Englesberg. 1967. Further evidence for positive control of the L-arabinose system by gene araC. J. Mol. Biol. 25:443-454. Shimada, K., R. Weisberg, and M. E. Gottesman. 1972. Prophage lambda at unusual chromosomal locations. I. Location of the secondary attachment sites and the properties of the lysogens. J. Mol. Biol. 63:483-503. Shimada, K., R. A. Weisberg, and M. E. Gottesman. 1973. Prophage lambda at unusual chromosomal locations. III. The components of the secondary attachment in Escherichia coli K-12. J. Mol. Biol. 80:297-314. Shimada, K., R. A. Weisberg, and M. E. Gottesman. 1975. Prophage lambda at unusual chromosomal locations. III. The components of the secondary attachment sites. J. Mol. Biol. 93:415-430. Szybalski, W., H. Kubinski, Z. Hradecna, and W. C. Summers. 1971. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 21, p. 383-413. Academic Press Inc., New York. Taylor, A. L., and C. D. Trotter. 1972. Linkage map of Escherichia coli K-12. Bacteriol. Rev. 36:504-524. Wilcox, G., J. Boulter, and N. Lee. 1974. The direction of transcription of the regulatory gene araC in Escherichia coli B/r. Proc. Natl. Acad. Sci. U.S.A. 71:3635-3639. Wilcox, G., J. Singer, and L. Heffernan. 1971. Deoxyribonucleic acid-ribonucleic acid hybridization studies on the L-arabinose operon of Escherichia coli B/r. J. Bacteriol. 108:1-4. Yarmolinsky, M. 1971. Making and joining DNA ends, p. 97-111. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Press, New York.