Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

Lactose utilization byVibrio vulnificus

The ability of wild-type strains ofVibrio vulnificus to utilize lactose as a sole source of carbon and energy and produce acid in lactose-containing media is associated with the appearance of spontaneous lactose-utilizing mutants. These contain increased activities of an enzyme able to hydrolyzeo-nitrophenyl--d-galactoside as well as lactose. This activity is constitutive in some mutants and inducible by both lactose and isopropyl--d-thiogalactoside in others. A limited survey of otherVibrio species indicates thatV. pelagius also can acquire, by mutation, the ability to grow on and make acid from lactose. No immunological cross-reaction was detected between the enzymes fromVibrio and the -galactosidases ofEscherichia coli andKlebsiella.     

Increased furan tolerance in Escherichia coli due to a cryptic ucpA gene

Expression arrays were used to identify 4 putative oxidoreductases that were upregulated (>3-fold) by furfural (15 mM, 15 min). Plasmid expression of one (ucpA) increased furan tolerance in ethanologenic strain LY180 and wild-type strain W. Deleting ucpA decreased furfural tolerance. Although the mechanism remains unknown, the cryptic ucpA gene is now associated with a phenotype: furan resistance.     

乳糖诱导甜蛋白Monellin在大肠杆菌中的表达

根据已报道的单链Monellin甜蛋白的氨基酸序列,按大肠杆菌基因偏爱密码子,设计和人工合成了单链monellin基因。将单链monellin基因克隆到大肠杆菌表达载体pET-28a中,构建了重组表达载体pET28a-mon,转化大肠杆菌BL21(DE3),得到表达Monellin的大肠杆菌工程菌株。借助SDS-PAGE分析方法,研究了乳糖代替IPTG诱导大肠杆菌表达甜蛋白Monellin。通过对乳糖作为诱导剂表达条件进行优化,Monellin的表达量可占细胞总蛋白的33.09%,与IPTG诱导表达量接近。本研究结果为乳糖作为诱导剂应用于重组大肠杆菌生产甜蛋白Monellin提供了参考依据。     

Effects of Dimethylsulfoxide on the Lactose Operon in Escherichia coli

Fowler, Audree V. (University of California, Los Angeles), and Irving Zabin. Effects of dimethylsulfoxide on the lactose operon in Escherichia coli. J. Bacteriol. 92:353-357. 1966.-Dimethylsulfoxide (DMSO) at a concentration of 5% (v/v) in the culture medium inhibits the growth of Escherichia coli to only a slight extent, and does not affect the differential rate of synthesis of beta-galactosidase. Resting cells remain viable after shaking in the presence of 20% DMSO for 3 hr at 37 C. Both beta-galactosidase and thiogalactoside transacetylase retain almost all activity after incubation in even higher concentrations of the solvent for many hours. DMSO decreases the permeability barrier. The rate of hydrolysis of o-nitrophenyl-beta-d-galactoside (ONPG) in whole cells containing beta-galactosidase but lacking permease is increased in cells treated with 5% DMSO. Several permeaseless strains preinduced for beta-galactosidase will grow on lactose in the presence, but not in the absence, of 5% DMSO. When permeaseless strains are grown on tetrazolium-lactose-agar, the presence of 5% DMSO causes a definite but not marked shift toward the lactose-positive character.     

Leptospira interrogans encodes an ROK family glucokinase involved in a cryptic glucose utilization pathway

Although Leptospira interrogans is unable to utilize glucose as its carbon/energy source, the LA_1437 gene of L. interrogans serovar Lai potentially encodes a group III glucokinase (GLK). The L. interrogans GLK (LiGLK) heterogeneously expressed in Escherichia coli was purified and proved to be a homodimeric enzyme with its specific activity of 12.3 ± 0.6 U/mg x protein determined under an improved assay condition (pH 9.0, 50 ° C), 7.5-fold higher than that assayed under the previously used condition (pH 7.3, 25 ° C). The improved sensitivity allowed us to detect this enzymatic activity of (5.0 ± 0.6) × 10(-3) U/mg x protein in the crude extract of L. interrogans serovar Lai cultured in standard Ellinghausen-McCullough-Johnson-Harris medium. The k(cat) and K(m) values for d-glucose and ATP were similar to those of other group III GLKs, although the K(m) value for ATP was slightly higher. Site-directed mutagenesis analysis targeting the conserved amino acid residues in the potential ATP-binding motif hinted that a proper array of Gly residues in the motif might be important for maintaining the conformation that was essential for its function. Gene expression profiling and quantitative proteomic data mining provided preliminary evidence for the absence of efficient systems involved in glucose transport and glycolysis that might account for the failure of glucose utilization in L. interrogans.     

Lactose permease of Escherichia coli catalyzes active beta-galactoside transport in a gram-positive bacterium.

The following several lines of evidence demonstrate that lactose permease (LacY) of Escherichia coli is assembled into the cytoplasmic membrane of gram-positive Corynebacterium glutamicum, expressing the lacY gene, as a functional carrier protein. (i) LacY was detected immunologically in the cytoplasmic membrane fraction of the heterologous host. (ii) Recombinant C. glutamicum cells bearing the lacY gene displayed an increased influx of o-nitrophenyl-beta-D-galactopyranoside, which was inhibited by N-ethylmaleimide. (iii) Washed cells were capable of accumulating methyl-beta-D-thiogalactoside about 60-fold. (iv) The uptake of methyl-beta-D-thiogalactoside was energy dependent and could be inhibited by the addition of 10 microM carbonyl cyanide-m-chlorophenylhydrazone. LacY of E. coli was active in the recombinant C. glutamicum cells despite the different membrane lipid compositions of these organisms.     

Asparagine utilization in Escherichia coli

Asparagine-requiring auxotrophs of Escherichia coli K-12 that have an active cytoplasmic asparaginase do not conserve asparagine supplements for use in protein synthesis. Asparagine molecules entering the cell in excess of the pool required for use of this amino acid in protein synthesis are rapidly degraded rather than accumulated. Supplements are conserved when asparagine degradation is inhibited by the asparagine analogue 5-diazo-4-oxo-l-norvaline (DONV) or mutation to cytoplasmic asparaginase deficiency. A strain deficient in cytoplasmic asparaginase required approximately 260 mumol of asparagine for the synthesis of 1 g of cellular protein. The cytoplasmic asparaginase (asparaginase I) is required for growth of cells when asparagine is the nitrogen source. This enzyme has an apparent K(m) for l-asparagine of 3.5 mM, and asparaginase activity is competitively inhibited by DONV with an apparent K(i) of 2 mM. The analogue provides a time-dependent, irreversible inhibition of cytoplasmic asparaginase activity in the absence of asparagine.     

Glucose and acetate influences on the behavior of the recombinant strainEscherichia coli HB 101 (GAPDH)

Summary This study highlights data about the production of a recombinant protein (glyceraldehyde-3-phosphate dehydrogenase) byE. coli HB 101 (GAPDH) during batch and fed-batch fermentations in a complex medium. From a small number of experiments, this strain has been characterized in terms of protein production performance and glucose and acetate influences on growth and recombinant protein production. The present results show that this strain is suitable for recombinant protein production, in fed-batch culture 55 g L^–1 of biomass and 6 g L^–1 of GAPDH are obtained. However this strain, and especially GAPDH overproduction is sensitive to glucose availability. During fermentations, maximum yields of GAPDH production have been obtained in batch experiments for glucose concentration of 10 g L^–1, and in fed-batch experiments for glucose availability of 10 g h^–1 (initial volume 1.5 L). The growth of the strain and GAPDH overproduction are also inhibited by acetate. Moreover acetate has been noted as an activator of its own formation.     

Lactose Variability of Escherichia coli in Thermally Stressed Reactor Effluent Waters

Lactose-utilizing and nalidixic acid-resistant populations of Escherichia coli, having an optimum growth temperature of 37°C, were placed in modified diffusion chambers. The chambers were submerged in the epilimnion and hypolimnion of a 1,100-hectare lake (Par Pond) which receives cooling water from a nuclear production reactor. Control chambers were placed in a deep-water reservoir and a Flowing-Streams Laboratory, both of which had comparable temperatures to Par Pond. The populations of E. coli were sampled regularly for up to 3 weeks. Viability of the bacteria was determined by dilution plating to nutrient agar followed by replicate plating onto selective medium to determine lactose utilization and nalidixic acid sensitivity. Initial populations of E. coli were lactose positive but changed to lactose negative in Par Pond when the reactor was operating (i.e., cooling water from the heat exchangers was being discharged to the lake). This alteration occurred most rapidly in the chambers closest to the cooling-water discharge point. Such changes did not occur in a deep-water reservoir, in Par Pond when the reactor was not operating, or in the Flowing-Streams Laboratory. The nalidixic acid-resistant characteristic remained stable regardless of the chambers' placement or reactor operations. Although the reasons for such alterations are unclear, it appears that lactose-negative populations of E. coli are selected for in these reactor effluent waters. The loss of the lactose characteristic prevents the recognition and identification of E. coli in this cooling lake (when the reactor is operating) and may prevent the assessment of water quality based on coliform recognition.