Isolation of mutants with impaired retroinhibition of histidine biosynthesis

The Escherichia coli, strain possessing purF, deoD and add mutations converts exogenous adenine into guanine nucleotides exclusively by the pathway coupled with histidine biosynthesis. When grown on adenine, this strain demonstrated sensitivity to histidine, thus making it possible to select histidine-resistant hisGR mutants with ATP-phosphoribosyltransferase desensibilized for histidine. The hisGR mutations were obtained in two his operons introduced into the his operon-sensitive E. coli strain: his operon of Salmonella typhimurium incorporated in DNA and his operon of E. coli on the F'episome. In both cases, the hisGR mutants obtained were shown to excrete histidine.     

Requirement for membrane potential in active transport of glutamine by Escherichia coli.

The effect of reducing the membrane potential on glutamine transport in cells of Escherichia coli has been investigated. Addition of valinomycin to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid-treated E. coli cells in the presence of 20 mM exogenous potassium reduced the membrane potential, as measured by the uptake of the lipophilic cation triphenylmethylphosphonium, and caused a complete inhibition of glutamine transport. Valinomycin plus potassium also caused a rapid decrease in the intracellular levels of ATP of normal E. coli cells, but had little if any effect on the ATP levels of two mutants of E. coli carrying lesions in the energy-transducing ATP complex (unc mutants). Yet both the membrane potential and the capacity to transport glutamine were depressed in the unc mutants by valinomycin and potassium. These findings are consistent with the hypothesis that both ATP and a membrane potential are essential to the active transport of glutamine by E. coli cells.     

Pathways for the incorporation of exogenous fatty acids into phosphatidylethanolamine in Escherichia coli

Two distinct pathways for the incorporation of exogenous fatty acids into phospholipids were identified in Escherichia coli. The predominant route originates with the activation of fatty acids by acyl-CoA synthetase followed by the distribution of the acyl moieties into all phospholipid classes via the sn-glycerol-3-phosphate acyltransferase reaction. This pathway was blocked in mutants (fadD) lacking acyl-CoA synthetase activity. In fadD strains, exogenous fatty acids were introduced exclusively into the 1-position of phosphatidylethanolamine. This secondary route is related to 1-position fatty acid turnover in phosphatidylethanolamine and proceeds via the acyl-acyl carrier protein synthetase/2-acylglycerophosphoethanolamine acyltransferase system. The turnover pathway exhibited a preference for saturated fatty acids, whereas the acyl-CoA synthetase-dependent pathway was less discriminating. Both pathways were inhibited in mutants (fadL) lacking the fatty acid permease, demonstrating that the fadL gene product translocates exogenous fatty acids to an intracellular pool accessible to both synthetases. These data demonstrate that acyl-CoA synthetase is not required for fatty acid transport in E. coli and that the metabolism of exogenous fatty acids is segregated from the metabolism of acyl-acyl carrier proteins derived from fatty acid biosynthesis.     

Regulation of bacterial glycogen synthesis. Stimulation of glycogen synthesis by endogenous and exogenous cyclic adenosine 3':5'-monophosphate in Escherichia coli and the requirement for a functional CRP gene

In Escherichia coli cya mutants, deficient in adenylate cyclase (EC 4.6.1.1), basal cellular rates of glycogen synthesis were lower and the relative increases produced by exogenous cyclic adenosine 3',5'-monophosphate during growth on glucose were greater than in their respective parent strains. These observations provide strong evidence that endogenous cyclic AMP is one of the key regulators of glycogen synthesis in growing E. coli. In crp mutants, deficient in cyclic AMP receptor protein (CRP), the basal cellular rates of glycogen synthesis were much lower than in their respective parent strains. Stimulation of glycogen synthesis by exogenous cyclic AMP was markedly attenuated in the three crp mutants. Thus, stimulation of glycogen synthesis by either endogenous or exogenous cyclic AMP appears to require CRP. Functional CRP appeared to be required for all three responses observed after cyclic AMP addition: an abrupt step-up in the cellular rate of glycogen synthesis, a continuing exponential increase in rate, and a stimulation of the rate during a subsequent nitrogen starvation. To account for these responses, we derived a mathematical model in which the cyclic AMP-CRP complex regulates the differential rate of synthesis of an enzyme metabolizing an effector of the rate-limiting enzyme of glycogen synthesis.     

Role of gene fadR in Escherichia coli acetate metabolism.

Mutants of Escherichia coli K-12 constitutive for fatty acid degradation (fadR) showed an increased rate of utilization of exogenous acetate. Acetate transport, oxidation, and incorporation into macromolecules was approximately fivefold greater in fadR mutants than fadR+ strains during growth on succinate as a carbon source. This effect was due to the elevated levels of glyoxylate shunt enzymes in fadR mutants, since (i) similar results were seen with mutants constitutive for the glyoxylate shunt enzymes (iclR), (ii) induction of the glyoxylate shunt in fadR+ strains by growth on acetate or oleate increased the rate of acetate utilization to levels comparable to those in fadR mutants, and (iii) fadR and fadR+ derivatives of mutants defective for the glyoxylate shunt enzymes showed equivalent rates of acetate utilization under these conditions. These results suggest that the operation of the glyoxylate shunt may play a significant role in the utilization of exogenous acetate by fadR mutants.     

Increasing the oxidative stress response allows Escherichia coli to overcome inhibitory effects of condensed tannins

Tannins are plant-derived polyphenols with antimicrobial effects. The mechanism of tannin toxicity towards Escherichia coli was determined by using an extract from Acacia mearnsii (Black wattle) as a source of condensed tannins (proanthocyanidins). E. coli growth was inhibited by tannins only when tannins were exposed to oxygen. Tannins auto-oxidize, and substantial hydrogen peroxide was generated when they were added to aerobic media. The addition of exogenous catalase permitted growth in tannin medium. E. coli mutants that lacked HPI, the major catalase, were especially sensitive to tannins, while oxyR mutants that constitutively overexpress antioxidant enzymes were resistant. A tannin-resistant mutant was isolated in which a promoter-region point mutation increased the level of HPI by 10-fold. Our results indicate that wattle condensed tannins are toxic to E. coli in aerobic medium primarily because they generate H(2)O(2). The oxidative stress response helps E. coli strains to overcome their inhibitory effect.     

Motility development of Salmonella typhimurium cells with flaV mutations after addition of exogenous flagellin

Cells of Salmonella typhimurium with flaV mutations developed motility after exogenous addition of flagellin, similar to what is observed with flbC mutants of Escherichia coli K-12.     

Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene.

Unlike enteric bacteria, Pseudomonas spp. generally lack thymidine phosphorylase and thymidine kinase activities, thus preventing their utilization of exogenous thymine or thymidine and precluding specific radioactive labeling of their DNA in vivo. To overcome this limitation, a DNA fragment encoding thymidine kinase (EC 2.7.1.21) from Escherichia coli was cloned into pKT230, a small, broad-host-range plasmid derived from plasmid RSF1010. From transformed E. coli colonies, the recombinant plasmid bearing the thymidine kinase gene was conjugally transferred to Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes. Thymidine kinase activity was expressed in all of these species, and all gained the ability to incorporate exogenous [2-14C]thymidine into their DNA. Thymidine incorporation into P. stutzeri was enhanced 12-fold more in mutants lacking thymidylate synthetase activity. These mutants produced higher levels of thymidine kinase and were thymidine auxotrophs; thymineless death resulted from removal of thymidine from a growing culture.     

Second-Site Revertants of Escherichia Coli Trp Repressor Mutants

Second-site reversion studies were performed with five missense mutants with defects in the trp repressor of Escherichia coli. These mutants were altered throughout the gene. The same unidirectional mutagen used in the isolation of these mutants, hydroxylamine, was used in reversion studies, to increase the liklihood that the revertants obtained would have second-site changes. Most of the second-site revertants were found to have the same amino acid substitutions detected previously as superrepressor changes. These second-site revertant repressors were more active in vivo than their parental mutant repressors, in the presence or absence of exogenous tryptophan. Apparently superrepressor changes at many locations in this protein can act globally to increase the activity of mutant repressors.     

Transketolase Mutants of Escherichia coli

Transketolase mutants have been selected after ethyl methane sulfonate mutagenesis of Escherichia coli. These strains are unable to grow on any pentose and, in addition, require a supplement of aromatic amino acids or shikimic acid for normal growth on any other carbon source. Revertants are normal in both respects and also contain transketolase. Transketolase mutants do not require exogenous pentose for growth. Preliminary genetic mapping of the locus is presented.     

The regulation and role of the periplasmic copper, zinc superoxide dismutase of Escherichia coli

The discovery of superoxide dismutase (CuZnSOD) within the periplasms of several Gram-negative pathogens suggested that this enzyme evolved to protect cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. However, its presence in some non-pathogenic bacteria implies that there may be a role for this SOD during normal growth conditions. We found that sodC, the gene that encodes the periplasmic SOD of Escherichia coli, is repressed anaerobically by Fnr and is among the many antioxidant genes that are induced in stationary phase by RpoS. Surprisingly, the entry of wild-type E. coli into stationary phase is accompanied by a several-hour-long period of acute sensitivity to hydrogen peroxide. Induction of the RpoS regulon helps to diminish that sensitivity. While mutants of E. coli and Salmonella typhimurium that lacked CuZnSOD were not detectably sensitive to exogenous superoxide, both were killed more rapidly than their parent strains by exogenous hydrogen peroxide in early stationary phase. This sensitivity required prior growth in air. Evidently, periplasmic superoxide is generated during stationary phase by endogenous metabolism and, if it is not scavenged by CuZnSOD, it causes an unknown lesion that augments or accelerates the damage done by peroxide. The molecular details await elucidation.     

FadD is required for utilization of endogenous fatty acids released from membrane lipids

FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth.     

Development of Auxotrophy by Streptomycin-Resistant Mutation

Several streptomycin-resistant mutants of Escherichia coli have been isolated which require exogenous isoleucine for growth. The majority of these strains were of streptomycin-dependent phenotype. If grown in the absence of streptomycin, these streptomycin-dependent auxotrophs (Sm(d-aux)) strains were unable to produce beta-galactosidase and aldolase activities and also failed to exhibit donor properties in conjugation. Genetic analysis indicated that the isoleucine requirement of these strains could be caused by a mutation at the strA locus.     

cobA function is required for both de novo cobalamin biosynthesis and assimilation of exogenous corrinoids in Salmonella typhimurium

Salmonella typhimurium is able to synthesize cobalamin (B12) under anaerobic growth conditions. The previously described cobalamin biosynthetic mutations (phenotypic classes CobI, CobII, and CobIII) map in three operons located near the his locus (minute 41). A new class of mutant (CobIV) defective in B12 biosynthesis was isolated and characterized. These mutations map between the cysB and trp loci (minute 34) and define a new genetic locus, cobA. The anaerobic phenotype of cobA mutants suggests an early block in corrin ring formation; mutants failed to synthesize cobalamin de novo but did so when the corrin ring is provided as cobyric acid dicyanide or as cobinamide dicyanide. Under aerobic conditions, cobA mutants were unable to convert either cobyric acid dicyanide or cobinamide dicyanide to cobalamin but could use adenosylcobyric acid or adenosylcobinamide as a precursor; this suggests that the mutants are unable to adenosylate exogenous corrinoids. To explain the anaerobic CobI phenotype of a cobA mutant, we propose that the cobA gene product catalyzes adenosylation of an early intermediate in the de novo B12 pathway and also adenosylates exogenous corrinoids. Under anaerobic conditions, a substitute function, known to be encoded in the main Cob operons, is induced; this substitute function can adenosylate exogenous cobyric acid and cobinamide but not the early biosynthetic intermediate. The cobA gene of S. typhimurium appears to be functionally equivalent to the btuR gene of Escherichia coli.     

Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli

Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).     

Analysis of ven3 and ven6 reticulate mutants reveals the importance of arginine biosynthesis in Arabidopsis leaf development

Arabidopsis thaliana reticulate mutants exhibit differential pigmentation of the veinal and interveinal leaf regions, a visible phenotype that often indicates impaired mesophyll development. We performed a metabolomic analysis of one ven6 (venosa6) and three ven3 reticulate mutants that revealed altered levels of arginine precursors, namely increased ornithine and reduced citrulline levels. In addition, the mutants were more sensitive than the wild-type to exogenous ornithine, and leaf reticulation and mesophyll defects of these mutants were completely rescued by exogenous citrulline. Taken together, these results indicate that ven3 and ven6 mutants experience a blockage of the conversion of ornithine into citrulline in the arginine pathway. Consistent with the participation of VEN3 and VEN6 in the same pathway, the morphological phenotype of ven3 ven6 double mutants was synergistic. Map-based cloning showed that the VEN3 and VEN6 genes encode subunits of Arabidopsis carbamoyl phosphate synthetase (CPS), which is assumed to be required for the conversion of ornithine into citrulline in arginine biosynthesis. Heterologous expression of the Arabidopsis VEN3 and VEN6 genes in a CPS-deficient Escherichia coli strain fully restored bacterial growth in minimal medium, demonstrating the enzymatic activity of the VEN3 and VEN6 proteins, and indicating a conserved role for CPS in these distinct and distant species. Detailed study of the reticulate leaf phenotype in the ven3 and ven6 mutants revealed that mesophyll development is highly sensitive to impaired arginine biosynthesis.     

Derivation of glycine from threonine in Escherichia coli K-12 mutants.

Escherichia coli AT2046 has been shown previously to lack the enzyme serine transhydroxymethylase and to require exogenous glycine for growth as a consequence. Strains JEV73 and JEV73R, mutants derived from strain AT2046, are shown here to be serine transhydroxymethylase deficient, but able to derive their glycine from endogenously synthesized threonine. Leucine is shown to be closely involved in the regulation of biosynthesis of glycine, to spare glycine in strain AT2046T, to replace glycine in strain JEV73, and to increase threonine conversion to glycine in a representative prototroph of E. coli. An interpretation of strains JEV73 and JEV73R as regulatory mutants of strain AT2046 is given. A hypothesis as to the role of leucine as a signal for nitrogen scavenging is suggested.     

Exogenous induction of the iron dicitrate transport system of Escherichia coli K-12.

Streptonigrin was used to select mutants impaired in the citrate-dependent iron transport system of Escherichia coli K-12. Mutants in fecA and fecB could not transport iron via citrate. fecA-lac and fecB-lac operon fusions were constructed with the aid of phage Mu dl(Ap lac). Strains deficient in ferric dicitrate transport which were mutated in fecB were as inducible as transport-active strains. They expressed the FecA outer membrane protein and beta-galactosidase of the fecB-lac operon fusions. In contrast, all fecA::lac mutants and fecA mutants induced with N-methyl-N'-nitro-N-nitrosoguanidine did not respond to ferric dicitrate supplied in the growth medium. tonB fecB mutants which were lacking all tonB-related functions were not inducible. We conclude that binding of iron in the presence of citrate to the outer membrane receptor protein is required for induction of the transport system. In addition, the tonB gene has to be active. However, iron and citrate must not be transported into the cytoplasm for the induction process. These data support our previous conclusion of an exogenous induction mechanism. Mutants in fur expressed the transport system nearly constitutively. In wild-type cells limiting the iron concentration in the medium enhanced the expression of the transport system. Thus, the citrate-dependent iron transport system shares regulatory devices with the other iron transport systems in E. coli and, in addition, requires ferric dicitrate for induction.     

小鼠RHOX5蛋白两种截短型突变体的原核表达及其与MDFIC互作的鉴定

目的：表达和纯化两种小鼠RHOX5蛋白的截短型突变体,确定完整的RHOX5蛋白及其两种截短型突变体与MDFIC蛋白结合的能力。方法：生物信息学分析小鼠同源异型框蛋白RHOX5的cDNA序列,分别对RHOX5的两种截短型片段RHOX5 N和RHOX5 C进行PCR、分别扩增RHOX5的两种截短型片段RHOX5 N和RHOX5 C并将其克隆至pGEX4T3原核表达载体,构建重组表达质粒。用重组表达质粒分别转化大肠杆菌RosettaTM2(DE3)菌株,经IPTG诱导后,使用Glutathione-Sepherase 4B颗粒对融合蛋白进行小批量亲和纯化,通过SDS-PAGE电泳分离目标蛋白,确定融合蛋白的表达。进行GST-pull down实验,检测完整的RHOX5蛋白及其两种截短型突变体与MDFIC蛋白结合的能力。 结果：在大肠杆菌RosettaTM2(DE3)中有效地实现了GST-RHOX5、GST-RHOX5 N和GST-RHOX5-C-3种融合蛋白的可溶性表达;经Glutathione Sepherase 4B颗粒亲和纯化后,获得了纯化后GST融合蛋白;GST-pull down实验证实,含有homeodomain的RHOX5蛋白和RHOX5C截短型突变体可以与MDFIC蛋白相结合,而RHOX5N截短型突变体则丧失了与MDFIC蛋白结合的能力。 结论：实现了RHOX5及其两种截短型突变体的原核表达和纯化,证实RHOX5蛋白的homeodomain结构域是其与MDFIC结合的关键部位。     

Isolation and characterization of Escherichia coli K-12 mutants lacking both 2-acyl-glycerophosphoethanolamine acyltransferase and acyl-acyl carrier protein synthetase activity.

2-Acyl-glycerophosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthetase are thought to be dual catalytic activities of a single inner membrane enzyme. A filter disc replica print method for the detection of acyl-ACP synthetase activity by colony fluorography was used to screen a mutagenized population of cells for acyl-ACP synthetase mutants (aas). All aas mutants lacked both acyl-ACP synthetase and 2-acyl-GPE acyltransferase activities in vitro. There was no detectable acyl-CoA-independent incorporation of exogenous fatty acids into phosphatidylethanolamine or the major outer membrane lipoprotein in aas mutants. Exogenous lysophospholipid uptake and acylation was also lacking in aas mutants. Lipoprotein acylation by phospholipids synthesized by the de novo biosynthetic pathway was not affected in aas mutants showing that this gene product was not directly involved in lipoprotein biogenesis. The aas mutants had an altered membrane phospholipid composition and accumulated both 2-acyl-GPE and acylphosphatidylglycerol. Acylphosphatidylglycerol accumulation was due to the transacylase activity of lysophospholipase L2 (the pldB gene product) since aas pldB double mutants accumulated 2-acyl-GPE, but not acylphosphatidylglycerol. The aas allele was mapped to 61 min of the Escherichia coli chromosome, and the deduced gene order in this region was thyA-aas-lysA. The biochemical, physiological, and genetic analyses of aas mutants support the conclusion that 2-acyl-GPE acyltransferase and acyl-ACP synthetase are two activities of the same protein and confirm that this enzyme system participates in membrane phospholipid turnover and governs the acyl-CoA independent incorporation of exogenous fatty acids and lysophospholipids into the membrane.