Escherichia coli B/r leuK mutant lacking pseudouridine synthase I activity.

Escherichia coli B/r strain EB146 containing mutation leuK16 has elevated levels of enzymes involved in the synthesis of leucine, valine, isoleucine, histidine, and tryptophan (Brown et al., J. Bacteriol. 135:542-550, 1978). We show here that strain EB146 (leuK16) has properties that are similar to those of E. coli and Salmonella typhimurium hisT strains. In tRNA1Leu from both hisT and leuK strains, positions 39 and 41 are uridine residues rather than pseudouridine residues. Furthermore, in tRNA3Leu and tRNA4Leu from a leuK strain, uridine residues at positions 39 and 40, respectively, are unmodified. Pseudouridine synthase I activity is missing in extracts of strain EB146 (leuK16), and extracts of strain EB146 (leuK16) and of a hisT strain do not complement one another in vitro. Four phenotypes of strain EB146 (leuK16), leucine excretion, wrinkled colony morphology, and elevated levels of leu and his enzymes, are complemented by a plasmid having a 1.65-kilobase DNA fragment containing the E. coli K-12 hisT locus. These results indicate that either leuK codes for pseudouridine synthase I (and is thus a hisT locus in reality) or, less likely, it codes for a product that affects the synthesis or activity of pseudouridine synthase I.     

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.     

trans-Recessive mutation in the first structural gene of the histidine operon that results in constitutive expression of the operon.

The first enzyme for histidine biosynthesis, encoded in the hisG gene, is involved in regulation of expression of the histidine operon in Salmonella typhimurium. The studies reported here concern the question of how expression of the histidine operon is affected by a mutation in the hisG gene that alters the allosteric site of the first enzyme for histidine biosynthesis, rendering the enzyme completely resistant to inhibition by histidine. The intracellular concentrations of the enzymes encoded in the histidine operon in a strain carrying such a mutation on an episome and missing the chromosomal hisG gene are three- to fourfold higher than in a strain carrying a wild-type hisG gene on the episome. The histidine operon on such a strain fails to derepress in response to histidine limitation and fails to repress in response to excess histidine. Furthermore, utilizing other merodiploid strains, we demonstrate that the wild-type hisG gene is trans dominant to the mutant allele with respect to this regulatory phenomenon. Examination of the regulation of the histidine operon in strains carrying the feedback-resistant mutation in an episome and hisT and hisW mutations in the chromosome showed that the hisG regulatory mutation is epistatic to the hisT and hisW mutations. These data provide additional evidence that the first enzyme for histidine biosynthesis is involved in autogenous regulation of expression of the histidine operon.     

Capsule synthesis by Bacillus anthracis is required for dissemination in murine inhalation anthrax

Bacillus anthracis, the agent of anthrax, produces a poly-D-glutamic acid capsule that has been implicated in virulence. Many strains missing pXO2 (96 kb), which harbors the capsule biosynthetic operon capBCAD, but carrying pXO1 (182 kb) that harbors the anthrax toxin genes, are attenuated in animal models. Also, noncapsulated strains are readily phagocytosed by macrophage cell lines, whereas capsulated strains are resistant to phagocytosis. We show that a strain carrying both virulence plasmids but deleted specifically for capBCAD is highly attenuated in a mouse model for inhalation anthrax. The parent strain and capsule mutant initiated germination in the lungs, but the capsule mutant did not disseminate to the spleen. A mutant harboring capBCAD but deleted for the cap regulators acpA and acpB was also significantly attenuated, in agreement with the capsule-negative phenotype during in vitro growth. Surprisingly, an acpB mutant, but not an acpA mutant, displayed an elevated LD(50) and reduced ability to disseminate, indicating that acpA and acpB are not true functional homologs and that acpB may play a larger role in virulence than originally suspected.     

Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits.

A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-F0 adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the F0 portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional F0 might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight F0 subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight F0 subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the alpha- and beta-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight F0 subunit and to the formation of a functional F0. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the F1-F0 ATPase complex to be proposed.     

Evidence for an Altered Operator Specificity: Catabolite Repression Control of the Leucine Operon in Salmonella typhimurium

A mutation, GD-1, in the leucine operon imposed unusual growth characteristics upon a leucine auxotrophic strain bearing the leucine operator mutation, leu-500. The strain with the GD-1 mutation was able to grow on a minimal salts medium when citrate was the sole carbon source, but required leucine when glucose was present. Tests with a large number of carbohydrates suggest that in the strain bearing the GD-1 mutation the leucine biosynthetic enzymes are under catabolite repressor control. Recombination studies indicate that the GD-1 mutation is a secondary alteration of the leucine operator at or very close to the site of the leu-500 mutation. Mutations at the supX locus (previously termed su leu 500 and located on the chromosome between the cysteine B and tryptophan gene clusters) result in elimination of the catabolite repression effect. The data are interpreted as an indication that the GD-1 and leu-500 mutations alter the leucine operator with respect to its specificity of response to repressors.     

A Fifth Gene (uncE) in the Operon Concerned with Oxidative Phosphorylation in Escherichia coli

Three mutant unc alleles (unc-408, unc-410, and unc-429) affecting the coupling of electron transport to oxidative phosphorylation in Escherichia coli K-12 have been characterized. Genetic complementation analyses using previously defined mutant unc alleles indicated that the new mutant unc alleles affect a previously undescribed gene designated uncE. The phenotype of strains carrying the uncE408 or uncE429 allele is similar in that Mg(2+)-adenosine triphosphatase activity is only found in the cytoplasmic fraction, and membranes do not bind the F(1) portion of adenosine triphosphatase purified from a normal strain. In contrast, adenosine triphosphatase activity is present both in the cytoplasm and on the membranes from a strain carrying the unc-410 allele, and normal F(1) binds to F(1)-depleted membranes from this strain. The adenosine triphosphatase solubilized from membranes of a strain carrying the unc-410 allele reconstituted ATP-dependent membrane energization in F(1)-depleted membranes from a normal strain. Genetic complementation tests using various Mu-induced unc alleles in partial diploid strains show that the uncE gene is in the unc operon and that the order of genes is uncB E A D C. The unc-410 allele differs from the uncE408 and uncE429 alleles in that complementation tests with the Mu-induced unc alleles indicate that more than one gene is affected. It is concluded that this is due to a deletion which includes part of the uncE gene and another gene, or genes, between the uncE and uncA genes.     

New cysE-pyrE-linked rfa mutation in Escherichia coli K-12 that results in a heptoseless lipopolysaccharide.

A new novobiocin-supersensitive mutant of Escherichia coli K-12 has been characterized biochemically and genetically. Lipopolysaccharide prepared from this mutant strain is truncated and contains 2-keto-3-deoxyoctulosonic acid as its only core sugar. This new core-defective mutation, designated rfa-2, results in increased sensitivity to several hydrophobic and some hydrophilic agents. Genetic analysis of the rfa mutant indicated that the rfa-2 locus is located at 81 min on the chromosome. The order of the genes in this region based on transduction analysis is xyl cysE rfa-2 rfaD70 pyrE. P1 transduction analyses indicate that the rfa-2 marker is nonallelic with the recently described cysE-pyrE-linked rfaD70 locus. Plasmids carrying the wild-type rfaD70+ allele failed to abolish the rfa-2 phenotypes. Further, the rfaD gene product, ADP-L-glycero-D-mannoheptose-6-epimerase, was detected in crude extracts of a rfa-2 mutant strain, CL609, and was absent in the rfaD70 mutant. The wild-type rfa-2 allele codes either for a specific heptose biosynthetic enzyme (different from the rfaD gene product) or an enzymatic activity required for the addition of heptose to the lipid A-2-keto-3-deoxyoctulosonic acid acceptor.     

The ilvIH operon of Escherichia coli is positively regulated.

The ilvIH operon of Escherichia coli (located near min 2) encodes acetohydroxyacid synthase III, an isozyme involved in branched-chain amino acid biosynthesis. A strain with lacZ fused to the ilvIH promoter was constructed. Transposon Tn10 was introduced into this strain, and tetracycline-resistant derivatives were screened for those in which ilvIH promoter expression was markedly reduced. In one such derivative, strain CV1008, beta-galactosidase expression was reduced more than 30-fold. The transposon giving rise to this phenotype inserted near min 20 on the E. coli chromosome. Extract from a wild-type strain contains a protein, the IHB protein, that binds to two sites upstream of the ilvIH promoter (E. Ricca, D. A. Aker, and J. M. Calvo, J. Bacteriol. 171:1658-1664, 1989). Extract from strain CV1008 lacks IHB-binding activity. These results indicate that the IHB protein is a positive regulator of ilvIH operon expression. The gene that encodes the IHB protein, ihb, was cloned by complementing the transposon-induced mutation. Definitive evidence that the cloned DNA encodes the IHB protein was provided by determining the sequence of more than 17 amino acids at the N terminus of the IHB protein and comparing it with the nucleotide sequence. A mutation that prevents repression of the ilvIH operon by leucine in vivo and that alters the DNA-binding characteristics of the IHB protein in vitro was shown to be an allele of the ihb gene. The ihb gene is identical to oppI, a gene that regulates the oppABCDF operon (E. A. Austin, J. C. Andrews, and S. A. Short, Abstr. Mol. Genet. Bacteria Phages, p. 153, 1989). Thus, oppI/ihb encodes a protein that regulates both ilvIH, an operon that is repressed by leucine, and oppABCDF, an operon involved in peptide transport that is induced by leucine. We propose that the designation lrp be used in the future instead of oppI or ihb and that Lrp (leucine-responsive regulatory protein) be used in place of IHB.     

Genomics of pyrrolnitrin biosynthetic loci: evidence for conservation and whole-operon mobility within gram-negative bacteria

Pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite produced by a narrow range of Gram-negative bacteria. The PRN biosynthesis by rhizobacteria presumably has a key role in their life strategies and in the biocontrol of plant diseases. The biosynthetic operon that encodes the pathway that converts tryptophan to PRN is composed of four genes, prnA through D , whose diversity, genomic context and spread over bacterial genomes are poorly understood. Therefore, we launched an endeavour aimed at retrieving, by in vitro and in silico means, diverse bacteria carrying the prnABCD biosynthetic loci in their genomes. Analysis of polymorphisms of the prnD gene sequences revealed a high level of conservation between Burkholderia , Pseudomonas and Serratia spp. derived sequences. Whole-operon- and prnD -based phylogeny resulted in tree topologies that are incongruent with the taxonomic status of the evaluated strains as predicted by 16S rRNA gene phylogeny. The genomic composition of c . 20 kb DNA fragments containg the PRN operon varied in different strains. Highly conserved and distinct transposase-encoding genes surrounding the PRN biosynthetic operons of Burkholderia pseudomallei strains were found. A prnABCD -deprived genomic region in B. pseudomallei strain K96243 contained the same gene composition as, and shared high homology with, the flanking regions of the PRN operon in B. pseudomallei strains 668, 1106a and 1710b. Our results strongly suggest that the PRN biosynthetic operon is mobile. The extent, frequency and promiscuity of this mobility remain to be understood.     

The GCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces cerevisiae.

The intracellular concentrations of the polypeptides encoded by the two enolase (ENO1 and ENO2) and three glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2, and TDH3) genes were coordinately reduced more than 20-fold in a Saccharomyces cerevisiae strain carrying the gcr1-1 mutation. The steady-state concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA was shown to be approximately 50-fold reduced in the mutant strain. Overexpression of enolase and glyceraldehyde-3-phosphate dehydrogenase in strains carrying multiple copies of either ENO1 or TDH3 was reduced more than 50-fold in strains carrying the gcr1-1 mutation. These results demonstrated that the GCR1 gene encodes a trans-acting factor which is required for efficient and coordinate expression of these glycolytic gene families. The GCR1 gene and the gcr1-1 mutant allele were cloned and sequenced. GCR1 encodes a predicted 844-amino-acid polypeptide; the gcr1-1 allele contains a 1-base-pair insertion mutation at codon 304. A null mutant carrying a deletion of 90% of the GCR1 coding sequence and a URA3 gene insertion was constructed by gene replacement. The phenotype of a strain carrying this null mutation was identical to that of the gcr1-1 mutant strain.     

Effect of alteration of the acetic acid synthesis pathway on the fermentation pattern of escherichia coli

The glucose metabolism of an Escherichia coli strain bearing mutations abolishing both acetyl phosphotransferase (PTA) and acetate kinase (ACK) activities was studied under aerobic and anaerobic conditions. These studies were conducted in a complex medium with the mutant carrying no plasmid, the mutant carrying the common cloning vector pUC19, and the mutant carrying a plasmid bearing the "pet" operon that encodes Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase activities. The mutant carrying no plasmid showed lower specific growth and glucose uptake rates relative to the parent wild-type strain (K-12), Lactic acid was produced at higher levels than the wild type, and considerable amounts of pyruvic acid were secreted as an unusual byproduct. Analysis of other fermentation products showed low but significant amounts of acetic acid, no accumulation of formic acid, and lower secretion of succinate and ethanol. The maintenance of the plasmid pUC19 in the mutant negatively affected metabolism. Expression of the pet operon overcame the metabolic stress caused by the plasmid, enhancing growth and glucose uptake rates to the values observed in the plasmidfree mutant. Also, expression of the pet operon allowed consumption of pyruvate accumulated during the first hours of fermentation.