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The pathway for histidinle biosyinthesis in Salmonella typhimurium, elucidated largely through the efforts o:f Dr. B. N. Ames and co-workers, 1-5 is shown in Figure 1. The figure also shows that portioin of the pathway for puriine biosynthesis which involves the further metabolism of 4-amino-5-imidazole carboxamide riboinucleotide (phosphoribosyl-AIC).6 Dr. Phillip Hartmain and his colleagues have shown that the structural genes for the enzymes catalyziing the reactioins of the histidine pathway are localized in a small segment of the bacterial chromosome, the histidine operon.7' 8 The phenomenoni of coordiniate derepressioin of the histidine enzymes was originally described by Ames and Garry, 9 who found that the histidine operon functions as a single unit in response to the level of histidiine available to the organism. Evidence presented by Martin10 inidicates that the histidinie operon is transcribed into a polycistroiiic messenger-RNA, anid studies oIn polarity in the histidine operon7' 11,12 suggest that this messenger-RNA is trainslated from the end which

Kinetic studies on the derepression of 5 of the 10 enzymes for histidine biosynthesis in Salmonnella typhimurium reveal that derepression may proceed by two different modes, depending upon the site of mutation in the various histidine auxotrophs examined. Simultaneous derepression of the enzymes is observed in those mutants incapable of producing the intermediate, 4-amino-5-imidazolecarboxamide ribonucleotide, through the histidine pathway. Derepression of the enzymes in a temporal sequence corresponding to the positional sequence of the genes in the histidine operon is observed in those mutants which do produce this intermediate. The addition of 4-amino-5-imidazolecarboxamide ribonucleoside to cultures of mutants ordinarily characterized by the simultaneous mode of derepression served to shift the mode from simultaneous to sequential, but did not influence the derepressed rate of enzyme synthesis. In organisms in which the sequential mode is ordinarily observed, adenine effected a shift to the simultaneous mode, causing all the enzymes to derepress at the same time, but did not influence the derepressed rate of enzyme synthesis. Chloramphenicol, at low concentrations, reduced the growth rate of derepressed cells by a factor of 2 to 3 and decreased the rate of derepression of the histidine enzymes in all organisms tested. In the histidine auxotroph, hisE11, and in the wild type, LT-2 (derepressed by the addition of thiazole alanine), chloramphenicol prolonged the intervals between the times of derepression of the histidine enzymes. On the other hand, in hisG52, in which the enzymes for histidine biosynthesis derepress simultaneously and not sequentially, chloramphenicol did not alter the time of derepression of the enzymes. In the presence of chloramphenicol, adenine shifted the mode of derepression of hisE11 and LT-2 from sequential to simultaneous, just as it did in the absence of chloramphenicol. However, adenine did not eliminate the effect of chloramphenicol on the rate of depression. The data presented here suggest that the histidine operon is transcribed into a polycistronic message which, under conditions in which sequential derepression is observed, is translated from one end only. Under conditions in which simultaneous derepression is observed, the translation of this message is initiated at multiple sites.

Kinetic studies on repression of the enzymes for histidine biosynthesis in Salmonella typhimurium showed that, upon addition of histidine to a derepressed culture, the enzymes became repressed in a temporal sequence which corresponds with the positional sequence of the genes in the histidine operon. This serial pattern of repression occurred under conditions in which the feedback site of the first enzyme for histidine biosynthesis is intact. When this site was rendered nonfunctional the pattern of repression was changed so that all of the enzymes became repressed concomitantly. These results suggest that the first enzyme for histidine biosynthesis plays a hitherto unrecognized role in control of the histidine system.

It would appear from these studies that nitrogen control reflects the catabolic capacity of the cell and that utilizable nitrogen sources and some carbon sources are, to some extent, in competition for this capacity. The series of catabolic events initiated by addition of D-amino acids or by growth on aldol sugars, in the presence of ammonia nitrogen in the growth medium, provide an opportunity for study of the positive aspect of nitrogen control under conditions where negative control predominates. This approach may eventually clarify the apparent interactions between the modification cascade components, PII and UT/UR, with the nitrogen regulatory gene, glnG. The utilization of nutrients by E. coli seems less a matter of energy than of expeditious use of whatever is offered in the diet. A comparison of the rate of increase of GS on cultural downshift with the rate of increase following D-glutamate addition would suggest that control by nitrogen limitation is about eight times more effective than positive activation by D-glutamate in the presence of ammonia nitrogen. This observation is consistent with the finding of an additive effect for the D-amino acids which can function as positive activators in GS regulation. It has been demonstrated for the wild-type organism that the increase in GS level generated by a mixture of D-glutamate, D-lysine, D-threonine, and glycine approximates the increase in GS level observed during step-down of the culture from an ammonia-sufficient to an ammonia-limited condition. This observation further supports the physiologic relevance of the effect of D-amino acids in nitrogen control and suggests that the apparent derepression of GS observed upon exhaustion of the ammonia nitrogen supply represents a composite of positive activation generated as alternative catabolic functions assume a greater importance. As might be expected, addition of D-glutamate to cells at the point of ammonia exhaustion had no additional positive effect. Following a downshift from glucose-ammonia-glutamine to glucose-glutamine cultural conditions, only the level of GS increases during the initial 60-minute observation period. This finding suggests that glutamine catabolism may, like D-threonine, D-lysine, and glycine, bypass the positive activation of GDH and GAT controls. The likely possibility in that the increases observed for GAT and GDH depend on D-glutamate as specific inducer. There are several instances where D-amino acids function as inducers of L-amino acid dehydrogenases and where amino acid racemase activity is directly coupled to flavoprotein dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)

The techniques of double isotope labeling and specific hybridization have been used to determine the size of the mRNA transcribed from the histidine operon in Salmonella typhimurium under conditions of derepression. The results obtained by both techniques agree with the previous results of Martin3 which demonstrated that, in a constitutive mutant, the histidine mRNA is polycistronic. Previous studies revealed that derepression of the enzymes for histidine biosynthesis may proceed by either of two modes1,2. One mode is characterized by simultaneous derepression of all the enzymes, whereas the other mode is characterized by the derepression of the enzymes in a temporal sequence which corresponds with the positional sequence of genes in the histidine operon. Since the present studies show that the mRNA transcribed from the histidine operon is always polycistronic, regardless of the mode of derepression, we may rule out the possibility that the difference between the two modes reflects any difference in the size of the histidine mRNA. Rather, the difference must lie in the manner in which the polycistronic histidine mRNA is translated.

Previous workt with adeninie-requiring auxotrophic mutaiits of Salmonella typhimurium showed that a sequential blockade in the biosynthesis of adenylic acid was due to the genetic loss of the bifunctional enzyme, adenylosuccinase (adenylosuccinate-AMP lyase, EC 4.3.2.2). The phenotypic conisequence of this mutation was a specific growth requirement for adenine and the accumulation of the substrate of the first reaction, succinyl-AICAR. By transductioni techniques the structural gene for this enzyme was localized on the Salmonella chromosome in a region designated as the pur-B locus. Subsequently, in vivo complementation was demonstrated among all the available pur-B mutants although none of these mutants showed any independent adenylosuccinase activity under the usual conditions of assay. 2 A new class of pur-B mutants with a uniquely altered adenylosuccinase has recently been identified. Examination of crude extracts for adenylosuccinase activity showed that this class of pur-B mutants contained a specific activity which was approximately 1/5 the adenylosuccinase activity measured in extracts of the wild type. The growth pattern of the members of this group, as well as the variation of their adenylosuccinase activity with nutritional history, suggested an unusual repression effect. Comparisons between the wild-type and mutant adenylosuccinase revealed several differences in stability characteristics. Moreover, the failure to demonstrate any gene product, which was cross-reactive with the enzymeneutralizing antibody, in extracts prepared from mutant cells grown in excess adenine further suggested a repression concomitant with the mutant alteration. This communication will present the results of studies with Salmonella typhimurium, strain pur-91, a representative of the new class of pur-B mutants.