MATERIALS AND METHODS The isolation of plaque-forming , specialized transducing phages , Xphut , has been described ( 8 ) .
The Xphut phages used here carry the c1857 thermoinducible repressor allele and the Ssus7 lysis-defective allele , except for those used in the experiment summarized in Table 2 , which are S + .
The hut genes present on these phages are wild-type , except for hutQ222 ( see ref .
7 ) on Xphut36 in strain GS139 ( Table 2 ) and for hutH1 ( see ref .
10 ) on Xphut36 in the strains described in Table 5 .
Methods Microbiological culture and genetic procedures have been described ( 7-11 ) .
Strains lysogenic for XcI857 derivatives were grown at 320C .
For the determination of enzyme activities , cells were grown in a minimal-medium containing succinate as principal carbon source and ammonia as principal nitrogen source ( 11 ) .
For use with E. coli strains , the medium was supplemented with 1.0 g of trisodium-citrate monohydrate and 1.0 mg of thiamine hydrochloride per liter .
Cell-free extracts were prepared and assayed for enzyme activity as described ( 10 , 11 ) .
A modified procedure for the assay of H and G enzymes in suspensions of whole-cells was used .
Cells were harvested by centrifugation and washed once with one half the original volume of buffer ( potassium phosphate , pH 7.4 , 0.01 M , containing 5 mM 2-mercaptoethanol ) , and resuspended in one-tenth the original volume of the same buffer .
The assay procedures were as described ( 11 ) , except that CTAB ( hexadecyltrimethylammonium bromide ) was added to the assay mixture at a final concentration of 20 og/ml .
The cells were incubated at 370C in the CTAB-buffer mixture for 10-30 min before the reaction was started by the addition of substrate .
The values obtained were normalized for the number of cells in the assay by determination of the absor-bance at 277 nm of an aliquot of cells suspended in diethanol-amine * HCI buffer ( pH 9.4 , 0.067 M ) containing 20 Mg/ml of CTAB .
One A277 unit corresponds to approximately 0.05 mg of protein per ml .
Specific activities are defined in the tables .
RESULTS Evidence that the hutC gene product is a protein We show here that a mutation in the hutC gene is subject to amber suppression .
Strains bearing the hutC7 allele express both hut operons constitutively ( 10 ) .
To determine the susceptibility of this allele to amber suppression we constructed nearly isogenic strains by transferring F ' hut + and F ' hutC7 episomes from S. typhimurium to E. coli strain MS6040 ( suIII + ) and as control to E. coli strain MS6041 ( Su - ) .
The specific activity of H enzyme ( coded by the H gene ) § formed by these strains was measured after growth in the presence or absence of histidine as inducer .
All E. coli strains we have examined lack all hut genes and enzyme activities .
Therefore , all Hut enzyme activities in these hybrids from the S. typhimurium carried the F ' come genes on hut episome .
The critical data ( Table 1 ) are the specific activities of H enzyme produced by the Su-hutC7 strain ( GS171 ) and by § Symbols in roman type indicate enzymes or phenotype , while symbols in italic type indicate genes or genotype .
Histidine the suIII + hutC7 strain ( GS169 ) grown in the absence of inducer .
Under this condition , the suIII + strain produces only about 10-15 % as much enzyme as the nearly isogenic Su-strain .
Thus , the suppressor has restored activity to the C gene , which is then able to repress the H gene .
Since amber suppressors act at the level of translation ( 12 ) , this result indicates that the product of the C gene is a protein .
Evidence that the hutC gene product is a repressor We have shown that the C + allele is dominant to C7 and other constitutive alleles of the C gene ( 7 ) .
This observation strongly suggests that the product of the C + gene is an active molecule which represses the hut operons , and that the product of the C-gene is an inactive molecule lacking the ability to repress .
Another possible interpretation is that the C + gene product is an activator , which promotes expression of the hut operons only in the presence of inducer , while the C-gene product is The strains were constructed as described ( 7 ) by transferring F ' gal + hut + bio + ( from NE324 ) or F ' gal + hutC7 bio + ( from NE301 ) to MS6041 ( gal-Su - ) or to MS6040 ( gal-suIII + ) by selecting Gal + recombinants .
The cells were grown in succinate-ammonia minimal-medium to which was added 0.2 % ihistidine as inducer where indicated .
H enzyme activity was determined in whole-cell suspensions to which CTAB was added as described in Methods .
Specific activity is expressed as nanomoles of product formed per minute per Am tinit Strains NE2 and NE385 have been described ( 10 , 11 ) .
Strains NE421 to NE424 and NE426 are spontaneous mutants of NE385 selected for their ability to grow on glucose-histidine .
Strains NE513 and NE514 were obtained by transduction of NE404 ( hutM145 , R9 , bio-26 ) to Bio + with phage P376 grown on NE464 ( hutC7 , R9 ) , which was ultimately derived from strains NE2 ( hutR9 ) and NE7 ( hutC7 ) by transduction and episome transfer .
Procedures for the growth of cells and assay of enzymes , and specific activity units are given in Table 1 .
We propose that the M promoter mutations result in decreased levels of U and H enzyme by increasing the concentration of repressor .
If this interpretation is correct , restoration of high levels of enzymes should accompany abolition of repression .
That is , constitutive mutants should be found among pseudorevertants of M mutants selected for higher activities of U and H enzymes .
Likewise , strains made constitutive by transduction should have regained the ability to synthesize the enzymes at a high rate .
These expectations were verified as follows .
The hutM mutants discussed here were originally derived from strain NE2 , which carries a promoter mutation ( hutR9 ) rendering the UH operon partially resistant to catabolite-repression and thus allowing the organism to grow on a medium containing glucose as principal carbon source and histidine as sole nitrogen source ( 11 ) .
Unlike their parent NE2 ( M + , R9 ) , the mutants NE363 ( M139 , R9 ) and NE385 ( M145 , R9 ) grow poorly on glucose-histidine medium and are thus phenotypically R + .
That these mutants NE363 and NE385 are still genotypically R9 was demonstrated by recovering , through transduction into M+R + recipients , strains that had the original HutR-phenotype .
The HutR + phenotype of NE363 and NE385 presumably results from their decreased levels of U and H enzymes relative to the parental strain NE2 ( see Table 3 ) .
( For the experiments reported in Table 3 , we used nearly isogenic strains derived from the original mutants .
NE402 and NE445 are comparable to NE2 ; NE433 , to NE363 ; and NE446 , to NE385 .
) We examined 67 spontaneous mutants of strain NE385 ( M145 , R9 ) selected for their ability to grow well on glucosehistidine medium .
None were true revertants of M145 , since The strains were constructed as described ( 8 ) .
GS67 ( F ' hutHI ) was infected with phages carrying normal hutM or deleted hutM , class 9 ; the recombinant phages carrying hutHI were used to lysogenize strain MR134 ; into these lysogens we introduced F'hutC + U160 or F ' hutC7 , U25 episomes from strains NE479 and NE438 , respectively .
Procedures for the growth of cells and assay of enzymes and specific activity units are given in Table 1 .
all had retained the ability to grow on a medium containing histidine as sole source of carbon and nitrogen , the phenotype for which the hutM mutants had been selected .
Unlike strain NE385 , however , all of these pseudorevertants produced H enzyme constitutively , as determined by the qualitative spot assay ( 9 ) .
Five of these mutants were analyzed further ( see Table 4 ) .
Three strains-NE421 , NE422 , and NE424-have the HutC-phenotype .
In the non-inducing medium , they produce high activities of H enzyme ( right-hand operon ) and G enzyme ( left-hand operon ) .
Two other strains , NE423 and NE426 , have the phenotype expected of mutants with an operator-constitutive mutation in the right-hand operon .
They have a higher uninduced level of H enzyme ( righthand operon ) but not of G enzyme ( left-hand operon ) .
Under induced conditions , all of the mutants , like strain NE2 , produce about twice as much H enzyme as the parent strain NE385 .
We replaced by transduction the C + gene in an M mutant with the constitutive C7 allele .
The specific activities of H enzyme in two hutMl45 , C7 , R9 recombinants , strains NE513 and NE514 , are twice as high as that in the parent , NE385 ( M145 , R9 ) ( see Table 4 ) .
These results support the hypothesis that the mutation in the M promoter ( M145 ) has increased the concentration of repressor .
Deletions removing hutM and abolishing repression We can account for the increased concentration of repressor in the M promoter mutants by assuming that the C gene is part of the operon that has M as its promoter ( left-hand operon ) .
Deletions removing hutM should therefore abolish expression of the C gene .
We have described the isolation of X phut transducing phages that carry deletions which enter the hut region from the left ( 8 ) .
Of interest here are those phages that have lost the M promoter but have retained the C ( P , R ) QUH genes .
The extents of these deletions are shown in Fig. 1 .