Joseph M. Calvo

Leucine-induced dissociation of Escherichia coli Lrp hexadecamers to octamers.

Lrp is a global regulator of metabolism in Escherichia coli that helps cells respond to changes in environmental conditions. The action of Lrp as a transcriptional activator or repressor is sometimes affected when the medium contains exogenous leucine. In this study, we examined the thermodynamics of leucine binding to Lrp and to a leucine response mutant, Lrp-1, and leucine-induced dissociation of Lrp hexadecamer to leucine-bound octamer. The results of dynamic light-scattering and fluorescence measurements suggest that Lrp has two leucine-binding sites, one a high-affinity site and the other a low-affinity site that is coupled to the dissociation reaction. The Gibbs free energy change for leucine binding to the high-affinity site is about -7.0 kcal/mol. Binding of two leucine molecules to low-affinity sites on the hexadecamer or one leucine molecule to one octamer induces the dissociation of hexadecamer to leucine-bound octamer. The Gibbs free energy change for leucine binding to the low-affinity site was estimated to be in the range -4.66 to -5.03 kcal/mol for leucine binding to an octamer or -6.01 to -6.75 kcal/mol for leucine binding to a hexadecamer. The thermodynamic parameters derived from this study were used together with other data to estimate the distribution of free Lrp hexadecamer, octamer, leucine-bound hexadecamer, and leucine-bound octamer in cells. Mathematical modeling, employed to simulate modulation of Lrp action in response to growth conditions, gave results that are consistent with known patterns of Lrp action on different operons.

EXPRESSION AND REGULATION OF VITELLOGENIN MESSENGER RNA IN THE MOSQUITO, AEDES AEGYPTI

Vitellogenin (yolk protein) gene expression in the mosquito was investigated at the level of mRNA using a subcloned fragment (403-1c) of the vitellogenin DNA derived from an Aedes aegypti genomic library. Message appeared 1–3 hr after a blood meal, peaked at 36 hr and was rapidly degraded thereafter. Fluctuations in levels of 20-hydroxyecdysone after a blood meal coincided with accumulation of vitellogenin message. Blood-fed, decapitated females injected with 5 μg of 20-hydroxyecdysone accumulated up to 75% of the message found in blood-fed controls. Fat bodies from non-blood-fed females incubated with physiological levels of 20-hydroxyecdysone and the juvenile hormone analog methoprene contained twice as much vitellogenin message as those incubated with 20-hydroxyecdysone alone. Methoprene alone had no effect.

Control of leu operon expression in Escherichia coli by a transcription attenuation mechanism.

The DNA sequence of 306 base-pairs preceding and within the first structural gene of the leu operon of Escherichia coli, leuA, was determined. A prominent feature of this sequence is a signal for transcription termination. In vitro, transcription does terminate at this site, yielding a leader RNA of about 165 nucleotides. This leader RNA is potentially translatable into a peptide containing 28 amino acids, four of which are adjacent Leu residues. Several regions of base complementarity exist within the leader RNA. When the nucleotide sequences of the leu leaders of E. coli and Salmonella typhimurium are compared these segments are conserved while other segments vary appreciably. The highly conserved regions are those thought to play a functional role in attentuation control of leu operon expression.

Isolation of mosquito vitellogenin genes and induction of expression by 20-hydroxyecdysone

Abstract Vitellogenic female Aedes aegypti contain abundant, 6500 nucleotide long RNAs that are not present in males or non-vitellogenic females and which were presumed to encode vitellogenin (VG). Three clones that hybridized to cDNA made to poly(A + )RNA from vitelogenic females, but not to cDNA made to male RNA, were selected from a genomic library. DNA from each clone hybridized to the 6500 nucleotide RNA species. Restriction enzyme mapping suggests the clones represent three distinct genes. The two that have been characterized share an uninterrupted region of homology about 6.5 kb long. Part of the coding region of one of the cloned genes was inserted into an expression vector, and the resulting polypeptide reacted specifically with antibodies to vitellogenin, thus confirming that the clones contain VG genes. Using one of the cloned genes as a probe on northern hybridizations we found that injection of 20-hydroxyecdysone into non-blood-fed decapitated females stimulated vitellogenin gene expression. The response was much greater in blood-fed decapitated females than in non-blood-fed females.

Organization of Lrp-binding sites upstream of ilvlH in Salmonella typhimurium

Lrp, a major regulatory protein in Escherichia coli, controls the expression of numerous operons, including ilvlH. Lrp binds to six sites upstream of ilvlH, and Lrp binding is required for ilvlH expression. We show here that an Lrp‐like protein is also present in Salmonella typhimurium. This protein can bind both E. coli and S. typhimurium ilvlH DNA, as can E. coli Lrp. Methidiumpropyl‐EDTA footprinting studies were performed with purified E. coli Lrp and S. typhimurium ilvlH DNA. Six binding sites were defined, three of them being similar to corresponding sites in E. coli, and three being organized differently. A consensus derived from six S. typhimurium sites is compatible with that derived from a similar analysis of E. coli sequences.

Cloning of an EcoRI-Generated fragment of the leucine operon of Salmonella typhimurium

Recombinant plasmids carrying part of the leucine operon of Salmonella typhimurium were isolated following transformation of an Escherichia coli leucine auxotroph to prototrophy with a ligated mixture of EcoRI-treated Salmonella DNA and plasmid pSC101 DNA. Plasmids pCV11 and pCV13, containing a 3.4-10(6) dalton DNA fragment ligated to the vector, had the leu operon oriented in opposite directions. The orientation of the leu operon relative to plasmid genes was determined. The 3.4-10(6) dalton fragment was ligated in to the EcoRI site of plasmid pMB9 yielding plasmids pCV12 (orientation as in pCV11) and pCV14 (orientation as in pCV13). The results of enzyme assays and complementation tests indicated that these plasmids carry functional leuA, leuB, and leuC genes but not a functional leuD gene. Furthermore, the following results indicated that they have a functional leu control region and promoter. Expression of plasmid leu genes was markedly enhanced under conditions of leucine limitation whereas introduction of a leu promoter mutation into the operon oriented in either direction with respect to plasmid genes had a strong negative effect upon leu operon expression. Transcriptional readthrough from plasmid promoters, if it occurs at all, must be small in comparison with transcription initiated at the leu promoter. RNA was isolated from leucine auxotrophs grown under conditions of repression and derepression and from prototrophic strains derepressed for the leucine operon as a result of mutations in leuO, leuS, and flrB. The rate of synthesis of leu mRNA, measured by hybridization to plasmid pCV12 DNA, was proportional in each case to leu enzyme levels.

ilvIH Operon Expression in Escherichia coli Requires Lrp Binding to Two Distinct Regions of DNA

The leucine-responsive regulatory protein Lrp regulates the expression of a number of operons in Escherichia coli, including the ilvIH operon. Earlier in vitro experiments showed purified Lrp binding to two regions of DNA proximal to the ilvIH promoter, an upstream region (-260 to -190) and a downstream region (-150 to -40). The effect of mutations in these regions on ilvIH promoter expression in vivo led to the proposal that activation of transcription required Lrp binding to downstream sites 3, 4, 5, and 6. Binding of Lrp to upstream sites 1 and 2 seemed to enhance promoter expression but was not absolutely required (Q. Wang and J. M. Calvo, J. Mol. Biol. 229:306-318, 1993). Here we present data that require a reevaluation of the above conclusion. Constructs having either a deletion of DNA or a 100-bp substitution of DNA upstream of position -160 showed no ilvIH promoter activity in vivo. These results unambiguously establish that DNA at or upstream of position -160 is required for ilvIH promoter expression. Together with previous results, we conclude that Lrp bound at downstream sites is necessary but not sufficient for promoter activation. In addition, insertion of 4, 6, 8, or 10 bp between the upstream and downstream regions also resulted in a very strong reduction of in vivo promoter expression, even though the binding of Lrp in vitro was not greatly affected by these mutations. Closer inspection showed that the affinity of Lrp for the upstream region of all of these constructs was about the same but that Lrp bound to the downstream region of the wild-type construct with a higher degree of cooperativity than in the case of the others. These mutations may have reduced promoter activity in vivo by eliminating a binding site for some transcription factor other than Lrp. Alternatively, the small-addition mutations may have affected the geometry of these complexes, preventing either an interaction between Lrps bound at upstream and downstream sites (which might be necessary for promoter expression) or preventing the positioning of Lrp bound at upstream sites for productive interaction with the promoter.

Global versus Local Regulatory Roles for Lrp-Related Proteins: Haemophilus influenzae as a Case Study

Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, including asnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated with E. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein from Haemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having an lrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies with lrp(+) and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. coli cell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function.

Characterization of the 3' end of the leucine operon of Salmonella typhimurium.

SummaryThe nucleotide sequence of the leuD gene of Salmonella typhimurium and of the downstream flanking region are presented. S1 mapping experiments identified 3′ endpoints of leu mRNA 140 and 285 nucleotides downstream of the UAA stop codon of leuD mRNA. Experiments employing pulse-labeled RNA suggest that these endpoints result from transcription termination rather than RNA processing. Our results indicate that the organization of the 3′ non-translated region of the leu operon from S. typhimurium resembles that of the trp operon of Escherichia coli. Further, our results suggest that the leu operon of S. typhimurium does not contain structural genes other than those identified by genetic experiments, i.e. leu,A,B,C and D.

Regulation of dihydrofolate reductase synthesis in Escherichia coli

SummaryTwo clones from the Clarke-Carbon Escherichia coli colony bank were resistant to inhibition by trimethoprim, a potent inhibitor of dihydrofolate reductase. Both clones had elevated levels of dihydrofolate reductase. Furthermore, trimethoprim resistance and elevated enzyme levels were associated with ColE1 plasmids that carried DNA from the trkC ksgA pdxA region of the E. coli chromosome. Plasmid pLC1437a was shown by two criteria to carry the structural gene for dihydrofolate reductase: 1) A partial diploid containing plasmid pLC1437a produced a kinetically-recognizable dihydrofolate reductase that was not present in the parent haploid strain. 2) Plasmid pLC1437a coded for dihydrofolate reductase in vitro. A 1,000 base pair fragment of plasmid pLC1437a containing fol was used as a probe to measure fol mRNA in a mutant strain isolated by Sheldon and Brenner (Molec. gen. Genet. 147, 91–97, 1976). The mutation in this strain, which results in constitutively-high levels of dihydrofolate reductase and in the inability of the strain to grow at 42° C, is cis dominant (Sheldon and Brenner, 1976). The results of kinetic hybridization and pulse-labeling experiments indicated that the regulatory mutant produced elevated levels of dihydrofolate reductase in response to an increased rate of synthesis of fol mRNA.