Inhibition of Escherichia coli Biodegradative Threonine Dehydratase by Pyruvate

Pyruvate inhibits Escherichia coli K-12 biodegradative threonine dehydratase activity by a mechanism distinct from product inhibition by alpha-ketobutyrate and catabolite inactivation by intermediary metabolites.     

Role for Free Isoleucine or Glycyl-Leucine in the Repression of Threonine Deaminase in Escherichia coli

In Escherichia coli, the three branched-chain amino acid activating enzymes appear to be essential for multivalent repression of the isoleucine- and valine-forming enzymes. The results of experiments with a mutant, strain CU18, having an altered threonine deaminase, indicate that free isoleucine and some form of threonine deaminase (the product of the ilvA gene) are also involved in multivalent repression. This strain exhibits abnormally high derepressibility but normal repressibility of its ilv gene products, and its threonine deaminase is inhibited only by high concentrations of isoleucine. In strain CU18, the isoleucine analogue, thiaisoleucine, is incapable of replacing isoleucine in the multivalent repression of the ilv genes, whereas the analogue can fully replace the natural amino acid in repression in other strains examined. The dipeptide, glycyl-leucine, which, like isoleucine, is a heterotropic negative effector of threonine deaminase but is not a substrate for isoleucyl-transfer ribonucleic acid synthetase, can completely prevent the accumulation of threonine deaminase-forming potential during isoleucine starvation in strains with normal threonine deaminases. It can not, however, prevent such accumulation in strain CU18 or in other strains in which threonine deaminase is insensitive to any concentration of isoleucine.     

Induction of Adenosine Deaminase in Escherichia coli

Supplementing the salts-glucose medium of Escherichia coli with adenine initiates induction of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), growth inhibition, and an increased potential for the net deamination of adenine. The extent and duration of these events are proportional to the initial adenine concentration and are dependent upon adenylate pyrophosphorylase and repression of histidine biosynthesis for maximal expression. The conversion of adenine to hypoxanthine, though limited in rate, occurs concurrently with induction and accounts for the progressively decreasing rate of deaminase induction, since hypoxanthine is a relatively ineffective inducer. The subsequent decrease in deaminase activity is due to dilution by continued cell division and by enzyme inactivation which occurs during the late-log and early-stationary phases. The partially purified deaminase is labile to a number of environmental conditions, particularly to phosphate buffers of pH 6.8 or less. A disproportionately slow rate of adenine deamination by cells utilizing lactate permits a more prolonged period of induction and, consequently, a greater quantity of enzyme to be synthesized; cell division, but not enzyme inactivation, reduces enzyme concentration. The adenosine deaminases of Aerobacter aerogenes and Salmonella typhimurium are not inducible.     

Threonine tRNAs and their genes in Escherichia coli

Summary The subject of this study was the threonine isoacceptor family of tRNAs in Escherichia coli and the genes coding for them. The goal was to identify and map all the genes and to determine the relative contribution of each gene to the tRNA pool. The mapping experiments exploited gene-dosage effects in partially diploid strains; if a strain harboring a particular F episome overproduced a particular tRNA species, it could be concluded that the gene for that tRNA was located on the chromosomal segment carried by the F. Isoacceptor tRNAs were distinguished by column fractionation. It was found that there are three major threonine tRNA species that occur in roughly equal amounts. These are tRNA ₁ ^Thr , which is encoded by a gene in the distal region of the rrnD ribosomal RNA operon, and tRNA ₃ ^Thr and tRNA ₄ ^Thr , which come from genes in the cluster thrU tyrU glyT thrT at 89 min on the map. The relative abundances of the tRNA species roughly match the reported frequencies of the codons that they recognize in mRNA. Although the tRNA ₄ ^Thr has a mismatched base pair that raised questions about its biological activity, it was found to be functional at least with respect to recognition by the threonyl-tRNA synthetase. An apparent fourth gene affecting threonine tRNA has been identified and mapped at 6–8 min; it is here designated thrW. It may be a structural gene for a minor tRNA species, present in one-third the amount of each of the major species, and chromatographically indistinguishable from tRNA ₄ ^Thr .A preliminary report of most of this work has appeared previously (M.M. Comer, Abstr. Annu. Meet. Am. Soc. Microbiol. 1980, p. 109)     

Multivalent Induction of Biodegradative Threonine Deaminase

To determine the inducer(s) of the biodegradative threonine deaminase in Escherichia coli, the effects of various amino acids on the synthesis of this enzyme were investigated. The complex medium used hitherto for the enzyme induction can be completely replaced by a synthetic medium composed of 18 natural amino acids. In this synthetic medium, the omission of each of the seven amino acids threonine, serine, aspartic acid, methionine, valine, leucine, and arginine resulted in the greatest loss of enzyme formation. These seven amino acids did not significantly influence the uptake of other amino acids into the cells. Furthermore, they did not stimulate the conversion of inactive enzyme into an active form, since they did not affect the enzyme level in cells in which protein synthesis was inhibited by chloramphenicol. Threonine, serine, aspartic acid, and methionine failed to stimulate enzyme production in cells in which messenger ribonucleic acid synthesis was arrested by rifampin, whereas valine, leucine, and arginine stimulated enzyme synthesis under the same conditions. Therefore, the first four amino acids appear to act as inducers of the biodegradative threonine deaminase in E. coli and the last three amino acids appear to be amplifiers of enzyme production. The term "multivalent induction" has been proposed for this type of induction, i.e., enzyme induction only by the simultaneous presence of several amino acids.     

Biosynthetic Threonine Deaminase from Proteus morganii

Biosynthetic threonine deaminase was purified to an apparent homogeneous state from the cell extract of Proteus morganii, with an overall yield of 7.5%. The enzyme had a s⁰_20,w of 10.0 S, and the molecular weight was calculated to be approximately, 228,000. The molecular weight of a subunit of the enzyme was estimated to be 58,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seemed to have a tetrameric structure consisting of identical subunits. The enzyme had a marked yellow color with an absorption maximum at 415 nm and contained 2 mol of pyridoxal 5′-phosphate per mol. The threonine deaminase catalyzed the deamination of l-threonine, l-serine, l-cysteine and β-chloro-l-alanine. Km values for l-threonine and l-serine were 3.2 and 7.1 mm, respectively. The enzyme was not activated by AMP, ADP and ATP, but was inhibited by l-isoleucine. The Ki for l-isoleucine was 1.17 mm, and the inhibition was not recovered by l-valine. Treatment with mercuric chloride effectively protected the enzyme from inhibition by l-isoleucine.     

Contact-Dependent Growth Inhibition Causes Reversible Metabolic Downregulation in Escherichia coli

Contact-dependent growth inhibition (CDI) is a mechanism identified in Escherichia coli by which bacteria expressing two-partner secretion proteins encoded by cdiA and cdiB bind to BamA in the outer membranes of target cells and inhibit their growth. A third gene in the cluster, cdiI, encodes a small protein that is necessary and sufficient to confer immunity to CDI, thereby preventing cells expressing the cdiBA genes from inhibiting their own growth. In this study, the cdiI gene was placed under araBAD promoter control to modulate levels of the immunity protein and thereby induce CDI by removal of arabinose. This CDI autoinhibition system was used for metabolic analyses of a single population of E. coli cells undergoing CDI. Contact-inhibited cells showed altered cell morphology, including the presence of filaments. Notably, CDI was reversible, as evidenced by resumption of cell growth and normal cellular morphology following induction of the CdiI immunity protein. Recovery of cells from CDI also required an energy source. Cells undergoing CDI showed a significant, reversible downregulation of metabolic parameters, including aerobic respiration, proton motive force (Δp), and steady-state ATP levels. It is unclear whether the decrease in respiration and/or Δp is directly involved in growth inhibition, but a role for ATP in the CDI mechanism was ruled out using an atp mutant. Consistent with the observed decrease in Δp, the phage shock response was induced in cells undergoing CDI but not in recovering cells, based on analysis of levels of pspA mRNA.Intercellular communication mechanisms enable bacteria to coordinate biological phenomena such as DNA uptake, differentiation for fruiting body development, light production, and swarming (7, 8). These cell-to-cell interactions enable individual bacteria to form a multicellular community, such as in a biofilm on a solid surface, under specific environmental conditions (20, 52). Similarly to multicellular organisms, bacteria have signal transduction mechanisms to facilitate cellular cross talk, including two-component regulatory systems and other cell surface ligand-receptor interactions that control cellular processes.We previously described a cross talk phenomenon designated as contact-dependent growth inhibition (CDI) in which one bacterial isolate (CDI⁺) blocks the growth of another bacterium when mixed together (4). CDI requires two contiguous genes, cdiB and cdiA, which encode proteins that are in the two-partner secretion (TPS) family. Overlapping the stop codon of cdiA is a downstream open reading frame designated cdiI, which encodes a 79-amino-acid protein that provides immunity to growth inhibition from cells expressing cdiBA (4). Evidence strongly indicates that cell-to-cell contact is required for growth inhibition. First, separation of CDI⁺ inhibitor cells and target cells by a 0.4-μm nitrocellulose membrane blocked CDI, distinguishing CDI from the class of soluble bacterial growth inhibitors known as bacteriocins. Second, to address the possibility that a very short-lived bacteriocin-like molecule might be released from CDI⁺ inhibitor cells, we separated inhibitor-target cell aggregates by fluorescence-activated cell sorting. Target cells within aggregates with CDI⁺ inhibitor cells lost viability, as measured by growth on LB medium, more rapidly than did unbound target cells, supporting the conclusion that cell-to-cell contact is required for CDI (4).In our previous work, analysis of CDI was carried out with a bipartite system using Escherichia coli inhibitor cells containing cdiB, -A, and -I on a multicopy plasmid that constitutively expressed CDI activity, cocultured with E. coli K-12 target cells. Mixing inhibitor cells with E. coli K-12 target cells resulted in a 5- to 6-log decrease in target cell number after only 1 to 2 h (4). Because this bipartite CDI assay contains both inhibitor and target cells, monitoring of target cell growth in real time is not possible. Thus, we have not been able to determine if CDI is a reversible process or a nonreversible toxin-like system. This is important in assessing the role of CDI in the biology of E. coli as well as its potential role in many gram-negative bacteria, including uropathogenic E. coli, Burkholderia pseudomallei, and Yersinia pestis that contain genes with significant sequence identity to cdiB and cdiA (4).Recently, in collaboration with J. Malinverni and T. Silhavy (Princeton University), we showed that the target cell receptor for CDI is BamA, an essential outer membrane protein (OMP). Homologues of bamA are conserved in genomes from bacteria to mitochondria (3). BamA is a key component of the β-barrel assembly machine required for biogenesis of many other OMPs (34, 43, 53). Our results indicated that the BamA receptor facilitates CdiB/CdiA-dependent cell-to-cell binding and growth inhibition since antibodies to BamA blocked formation of inhibitor-target cell aggregates and CDI (3). The ligand for BamA is not known, but it seems probable that it is CdiA, which is at the surface of inhibitor cells (4), and may form a short 40- to 50-nm fiber, such as filamentous hemagglutinin in Bordetella pertussis, based on sequence similarities (42). AcrB is also required for sensitivity of target cells to CDI, which acts downstream of the BamA receptor (3). AcrB is a protein that exports small molecules, including drugs/antibiotics, through the inner membrane, with further transport through the outer membrane in conjunction with AcrA and TolC (47, 55, 56). This export machine requires proton motive force (Δp) as an energy source (46). Markedly, only BamA and AcrB, independent from their respective export machines, are required for CDI (3). Based on these results, we developed a model in which CdiA of inhibitor cells bind to BamA on target cells, transmitting a signal into target cells such as a CdiA peptide (Fig. ​(Fig.1A).1A). This signal could enter cells through an AcrB portal to interact with an unidentified cytosolic target. It is also possible that AcrB could play an indirect role in CDI-mediated growth inhibition.Open in a separate windowFIG. 1.Construction of a contact-dependent autoinhibition system in Escherichia coli. (A) CDI model. CdiB and CdiA (CdiBA) expressed by one cell (inhibitor cell) binds to BamA in the outer membrane (OM) of an adjacent cell (target cell) (3). A signal, such as a CdiA peptide, is transferred through BamA to AcrB located in the inner membrane (IM), causing inhibition of cell growth by an unknown mechanism. CDI is modulated at the cell surface by certain pili, including pyelonephritis-associated pili (4), and by colanic acid capsule (3) and inside cells by CdiI immunity protein (4). (B) Map of the CDI autoinhibition plasmid (pDAL728).Here, we describe the development of a CDI autoinhibition system in which growth inhibition is regulated by controlled expression of the CdiI immunity protein, enabling examination of a single population of cells undergoing CDI. Using this system, we show that cells undergoing CDI have significantly reduced cellular respiration, Δp, and steady-state ATP levels. Notably, this metabolic downregulation, as well as cellular growth inhibition, is reversible. To the best of our knowledge, this is the first report of a natural, reversible system controlling bacterial metabolism and growth.     

Threonine prevents derepression of pyridoxine synthesis in Escherichia coli B

After 40 min of pyridoxal starvation, pyridoxine phosphate oxidase-less mutants of Escherichia coli B derepressed pyridoxine biosynthesis 13-fold to a rate of 1.7 X 10(-9) mol/h per mg of cells. Threonine at 100 mg/liter prevented this derepression but did not affect the continued synthesis of pyridoxine. Neither serine nor branched-chain amino acids altered the threonine effect.     

Threonine analogue resistant mutants of Escherichia coli K-12

Summary Mutants of Escherichia coli K-12 resistant to a threonine analogue (-amino--hydroxy valeric acid) were predominantly resistant to ethionine and overproduced both threonine and methionine (2 mg/ml each). Novelty of the mutants is discussed.     

Effect of Shift-Down and Growth Inhibition on Phospholipid Metabolism of Escherichia coli

The metabolism of phospholipids of Escherichia coli was studied under conditions which inhibit various metabolic processes. Phospholipid synthesis and turnover were not inhibited by growth-inhibitory amounts of various antibiotics. Turnover of phosphatidylglycerol (PG) was inhibited by small amounts of dinitrophenol and by anaerobiosis. Turnover of phosphatidylethanolamine (PE), which is not detected in control cultures, was demonstrated under conditions of incipient lysis. When cells were shifted down from a rich to a poor medium, PE synthesis was inhibited, and incorporation of glycerol into the distal position of PG was stimulated. Under these conditions, turnover of the phosphate and the acylated glycerol moieties of PG was inhibited. Increased synthesis of PE was detected when filamentous cells were induced to make septa. The results indicate that PE synthesis is related to growth and cell division, whereas PG metabolism is related to other cell processes.     

Threonine inhibition of the aspartokinase--homoserine dehydrogenase I of Escherichia coli. Threonine binding studies.

Both activities of the aspartokinase--homoserine I (AK-HSD) of Escherichia coli are inhibited by threonine. Careful threonine binding studies have now been done which have allowed us to distinguish the various effects of threonine on the enzyme. The ultrafiltration technique of H. Paulus ((1969) Anal. Biochem. 32, 101) for measuring ligand binding was shown to be comparable with equilibrium dialysis techniques. Reduction in error by utilization of this procedure enabled us to obtain evidence for two different sets of threonine sites by direct binding studies. The binding data were mathematically consistent with two independent classes of threonine sites, each of which contained four sites per tetramer and had a Hill coefficient of about 2.3--2.5. KD for the second set of sites was five- to tenfold greater than the high affinity sites, depending upon conditions. The data now suggest that the sequential model for site--site interactions adequately describes the cooperativity of threonine binding to the high affinity set of sites.     

Threonine as a carbon source for Escherichia coli.

Threonine can be used aerobically as the sole source of carbon and energy by mutants of Escherichia coli K-12. The pathway used involves the conversion of threonine via threonine dehydrogenase to aminoketobutyric acid, which is further metabolized by aminoketobutyric acid ligase, forming acetyl coenzyme A and glycine. A strain devoid of serine transhydroxymethylase uses this pathway and excretes glycine as a waste product. Aminoketobutyric acid ligase activity was demonstrated after passage of crude extracts through Sephadex G100.     

Transport of Biosynthetic Intermediates: Homoserine and Threonine Uptake in Escherichia coli

Although amino acid transport has been extensively studied in bacteria during the past decade, little is known concerning the transport of those amino acids that are biosynthetic intermediates or have multiple fates within the cell. We have studied homoserine and threonine as examples of this phenomenon. Homoserine is transported by a single system which it shares with alanine, cysteine, isoleucine, leucine, phenylalanine, threonine, tyrosine, and valine. The evidence for this being the sole system for homoserine transport is (i) a linear double-reciprocal plot showing a homoserine K(m) of 9.6 x 10(-6) M, (ii) simultaneous reduction by 85% of homoserine and branched-chain amino acid uptake in a mutant selected for its inability to transport homoserine, and (iii) simultaneous reduction by 94% of the uptake of homoserine and the branched-chain amino acids by cells grown in millimolar leucine. Threonine, in addition to sharing the above system with homoserine, is transported by a second system shared with serine. The evidence for this second system consists of (i) incomplete inhibition of threonine uptake by any single amino acid, (ii) only 70% loss of threonine uptake in the mutant unable to transport homoserine, and (iii) only 40% reduction of threonine uptake when cells are grown in millimolar leucine. In this last case, the remaining threonine uptake can only be inhibited by serine and the inhibition is complete.     

Volatile Fatty Acids and the Inhibition of Escherichia coli Growth by Rumen Fluid

Concentrations of volatile fatty acids (VFA) normally found in bovine rumen fluid inhibited growth of Escherichia coli in Antibiotic Medium 3. Acetic, propionic, and butyric acids each produced growth inhibition which was markedly pH-dependent. Little inhibition was observed at pH 7.0, and inhibition increased with decreasing pH. A combination of 60 mumoles of acetate, 20 mumoles of propionate, and 15 mumoles of butyrate per ml gave 96, 69, and 2% inhibition at pH 6.0, 6.5, and 7.0, respectively. Rumen fluid (50%) gave 89 and 48% inhibition at pH 6.0 and 6.5, respectively, and growth stimulation (22%) at pH 7.0. Rumen fluid inhibitory activity was heat-stable, was not precipitated by 63% ethyl alcohol, and was lost by dialysis and by treatment with anion-exchange resins but not with cation-exchange resins. These results are consistent with the idea that VFA are the inhibitory substances in rumen fluid. Previous results which indicated that rumen fluid VFA did not inhibit E. coli growth were due to lack of careful control of the final pH of the growth medium. The E. coli strain used does not grow in rumen fluid alone at pH 7.0.     

Adenine Deaminase Activity of the yicP Gene Product of Escherichia coli

During previous work on deriving inosine-producing mutants of Escherichia coli, we observed that an excess of adenine added to the culture medium was quickly converted to hypoxanthine. This phenomenon was still apparent after disruption of the known adenosine deaminase gene (add) on the E. coli chromosome, suggesting that, like Bacillus subtilis, E. coli has an adenine deaminase. As the yicP gene of E. coli shares about 35% identity with the B. subtilis adenine deaminase gene (ade), we cloned yicP from the E. coli genome and developed a strain that overexpressed its product. The enzyme was purified from a cell extract of E. coli harboring a plasmid containing the cloned yicP gene, and had significant adenine deaminase [EC 3.5.4.2] activity. It was deduced to be a homodimer, each subunit having a molecular mass of 60 kDa. The enzyme required manganese ions as a cofactor, and adenine was its only substrate. Its optimum pH was 6.5-7.0 and its optimum temperature was 60°C. The apparent K_m for adenine was 0.8 mM.     

Growth Inhibition and Appearance of the Membranous Structure in Escherichia coli Infected with Bacteriophage fd

Thirty-six mutants of fd, a virus that infects but does not kill Escherichia coli, were isolated; 35 mutants were categorized into six complementation groups. Abortive infection with mutants in genes 1, 3, 4, 5, and 6, but not in gene 2, produced a cessation of host cell growth, generally linked to low burst size and to the formation of aberrant intracytoplasmic membranous structures. The membranous structure was studied during infection with various phage and hosts. Appearance of the membranous structure was linked specifically to incomplete phage maturation at the cell membrane, rather than solely to the inhibition of host cell growth or to infection with mutant phage, since (i) in one host, cell growth was inhibited, but no membranous structure developed; and (ii) when antibody against virus was added to cells infected with wild-type phage, phage extrusion was inhibited, cell growth stopped, and the membranous structure once again developed.