A microtiter plate-based system for the semiautomated growth and assay of bacterial cells for β-galactosidase activity

Menzel, Rolf

doi:10.1016/0003-2697(89)90391-6

Cited by 75 publications

(32 citation statements)

References 4 publications

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“…For assays of promoter-lac fusion expression, strains were grown either anaerobically or aerobically in LBK buffered at different pHs ranging from 5.5 to 9.0. The buffers used were HOMOPIPES at pH 5.0, MES at (Table 1) by using the microtiter plate method described previously (40,51,59).…”

Section: Methodsmentioning

confidence: 99%

pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

2004

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During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.pH response is important for growth and survival of Escherichia coli in an environment such as the human gastrointestinal tract, in which the pH fluctuates over the range from pH 6 to 8 (14, 18). The role of pH in gene expression in E. coli and related enteric bacteria has been studied extensively, but it has been studied largely under aerobic conditions (7,11,59,64,66; for reviews see references 15 and 54). Relatively few studies have addressed the relationship between pH and anaerobiosis, the predominant condition of enteric growth (2); the beststudied cases include anaerobic acid induction of amino acid decarboxylases (1, 6, 61) and a limited two-dimensional (2-D) gel study of protein profiles (7). However, enteric bacteria behave very differently under anaerobic and aerobic conditions; for example, in microarrays more than one-third of the genes expressed during aerobic growth are ...

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Section: Methodsmentioning

confidence: 99%

pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

2004

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“…/3-Galactosidase assay. /3-Galactosidase activity was assayed by a modification of previous methods (22,23). After incubation, the yeast culture samples were transferred to conical 0.6-ml RIA tubes (so that a multitip pipetter could be used throughout the remainder of the assay for efficiency and reproducibility) (Sarstedt, Inc., Newton, NC) and centrifuged at 4,000 g for 10 min at 4°C to pellet the cells.…”

Section: Methodsmentioning

confidence: 99%

Estrogen levels in childhood determined by an ultrasensitive recombinant cell bioassay.

Klein¹,

Baron²,

Colli³

et al. 1994

J. Clin. Invest.

352

225

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We hypothesized that estradiol levels are higher in prepubertal girls than in prepubertal boys and that this greater secretion of estradiol might drive the more rapid epiphyseal development and earlier puberty in girls. Since previous estradiol assays have lacked adequate sensitivity to test the hypothesis of higher estradiol levels in girls, we developed a new ultrasensitive assay to measure estrogen levels. The assay uses a strain of Saccharomyces cerevisiae genetically engineered for extreme sensitivity to estrogen. Yeast were transformed with plasmids encoding the human estrogen receptor and an estrogen-responsive promoter fused to the structural gene for f-galactosidase. Ether extracts of 0.8 ml of serum were incubated with yeast for 8 h and the 38-galactosidase response was used to determine estrogen bioactivity relative to estradiol standards prepared in charcoalstripped plasma. The assay was highly specific for estradiol with < 3 % cross-reactivity with estrone, estriol, or estradiol metabolites. The detection limit was < 0.02 pg/ml estradiol equivalents (100-fold lower than existing assays). Using this assay, we measured estrogen levels in 23 prepubertal boys (9.4±2.0 yr) and 21 prepubertal girls (7.7+1.9 [SD] yr). The estrogen level in girls, 0.6+0.6 pg/ml estradiol equivalents, was significantly greater than the level in boys, 0.08+0.2 pg/ml estradiol equivalents (P < 0.05). We conclude that the ultrasensitive recombinant cell bioassay for estrogen is approximately 100-fold more sensitive than previous estradiol assays, that estrogen levels are much lower prepubertally, in both sexes, than reported previously, and that prepubertal girls have 8-fold higher estrogen levels than prepubertal boys. (J. Clin. Invest. 1994Invest. . 94:2475Invest. -2480

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“…Extracts were prepared with glass beads and a Braun homogenizer as described previously (37). Assays were conducted with 96-well microtiter plates (33,35), and Northern blots. Total RNA was prepared from the frozen cell pellets as previously described (24).…”

Section: Methodsmentioning

confidence: 99%

Translation of the yeast transcriptional activator GCN4 is stimulated by purine limitation: implications for activation of the protein kinase GCN2.

Rolfes¹,

Hinnebusch²

1993

Mol. Cell. Biol.

124

113

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The transcriptional activator protein GCN4 is responsible for increased transcription of more than 30 different amino acid biosynthetic genes in response to starvation for a single amino acid. This induction depends on increased expression of GCN4 at the translational level. We show that starvation for purines also stimulates GCN4 translation by the same mechanism that operates in amino acid-starved cells, being dependent on short upstream open reading frames in the GCN4 mRNA leader, the phosphorylation site in the a subunit of eukaryotic translation initiation factor 2 (eIF-2a), the protein kinase GCN2, and translational activators of GCN4 encoded by GCNI and GCN3. Biochemical experiments show that eLF-2a is phosphorylated in response to purine starvation and that this reaction is completely dependent on GCN2. As expected, derepression of GCN4 in purine-starved cells leads to a substantial increase in HIS4 expression, one of the targets of GCN4 transcriptional activation. gcn mutants that are defective for derepression of amino acid biosynthetic enzymes also exhibit sensitivity to inhibitors of purine biosynthesis, suggesting that derepression of GCN4 is required for maximal expression of one or more purine biosynthetic genes under conditions of purine limitation. Analysis of mRNAs produced from the ADE4, ADES,7, ADE8, and ADEI genes indicates that GCN4 stimulates the expression of these genes under conditions of histidine starvation, and it appeared that ADE8 mRNA was also derepressed by GCN4 in purine-starved cells. Our results indicate that the general control response is more global than was previously imagined in terms ofthe type of nutrient starvation that elicits derepression of GCN4 as well as the range of target genes that depend on GCN4 for transcriptional activation.The general amino acid control response of Saccharomyces cerevisiae is a regulatory mechanism for the induction of more than 30 genes involved in 11 amino acid biosynthetic pathways (reviewed in references 22 and 23). When S. cerevisiae is starved for amino acids (23) or when the activity of tRNA synthetases is impaired (34, 43), expression of the transcriptional activator protein GCN4 is increased and GCN4 stimulates the transcription of amino acid biosynthetic genes which are subject to the general control.Expression of GCN4 is regulated in response to amino acid availability by both positive and negative regulators. When amino acids are abundant, four short open reading frames (uORFs) in the GCN4 mRNA leader act in cis to repress GCN4 expression at the translational level by preventing ribosomes scanning the leader from reaching the GCN4 AUG codon. According to a recently proposed model (1), ribosomes will translate the first uORF encountered (uORF1) and resume scanning. Under nonstarvation conditions, essentially all of these ribosomes will reinitiate translation at one of the remaining uORFs, uORF2 to uORF4, and fail to reinitiate translation again at the GCN4 start codon.Products of the SUI2, SUI3, and multiple GCD genes are trans-a...

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A microtiter plate-based system for the semiautomated growth and assay of bacterial cells for β-galactosidase activity

Cited by 75 publications

References 4 publications

pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

Estrogen levels in childhood determined by an ultrasensitive recombinant cell bioassay.

Translation of the yeast transcriptional activator GCN4 is stimulated by purine limitation: implications for activation of the protein kinase GCN2.

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