LacZ-promoter fusions: the effect of growth

Warner, Jessica B.; Lolkema, Juke S.

doi:10.1099/00221287-148-5-1241

Cited by 11 publications

(13 citation statements)

References 14 publications

(23 reference statements)

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“…2a). The pre-steady-state period represents the time required for the β-galactosidase expression level to equilibrate between the synthesis rate and the growth rate (Warner & Lolkema, 2002). A similar time dependence for β-galactosidase activity was observed when B. subtilis CM002 was grown in the presence of -isocitrate, but the level of expression appeared to be somewhat lower than that observed for citrate (Fig.…”

Section: Analysis Of Citm Expressionmentioning

confidence: 58%

“…2c). The differences in β-galactosidase activity levels in the exponential growth phase between B. subtilis CM002 cultures grown on citrate or -isocitrate and on cis-aconitate could not be explained by different growth rates (Warner & Lolkema, 2002), since these were not affected much by the different substrates. To exclude the possibility that the observed induction of β-galactosidase activity by cis-aconitate might be the result of the slow conversion (hydration) of this TCA cycle intermediate into citrate or isocitrate during the course of bacterial growth, CSE minimal medium containing cis-aconitate was pre-incubated for 24 h at 37 mC under continuous shaking before inoculation.…”

Section: Analysis Of Citm Expressionmentioning

confidence: 86%

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Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+–citrate transporter CitM

Warner

Lolkema

2002

Microbiology

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Section: Analysis Of Citm Expressionmentioning

confidence: 58%

Section: Analysis Of Citm Expressionmentioning

confidence: 86%

Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+–citrate transporter CitM

Warner

Lolkema

2002

Microbiology

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“…In conclusion, transcription of ptsH relative to that of crh was found to be three-to ninefold stronger when a PTS substrate is utilized (Table 2). Control experiments (see Materials and Methods) excluded the possibility that the different growth rates may affect the ␤-galactosidase activities (21).…”

Section: Resultsmentioning

confidence: 99%

Drastic Differences in Crh and HPr Synthesis Levels Reflect Their Different Impacts on Catabolite Repression in Bacillus subtilis

2004

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In Bacillus subtilis, carbon catabolite repression (CCR) of catabolic genes is mediated by ATP-dependent phosphorylation of HPr and Crh. Here we show that the different efficiencies with which these two proteins contribute to CCR may be due to the drastic differences in their synthesis rates under conditions that cause CCR.In Bacillus subtilis, carbon catabolite repression (CCR) of many catabolic genes is mediated by ATP-dependent phosphorylation of Ser-46 of HPr and of its homologue Crh (4). Although these two proteins exhibit high sequence identity (45%) and are both efficiently phosphorylated by the HPr kinase/phosphorylase, their contributions to CCR differ (4). P-Ser-Crh can only partly substitute for P-Ser-HPr in CCR, whereas P-Ser-HPr can completely substitute for P-Ser-Crh in this signal transduction pathway. In order to understand the different behaviors of these two proteins, we compared the expression levels of the corresponding genes, crh and ptsH (encoding HPr), in the presence of different carbon sources by using transcriptional and translational lacZ reporter gene fusions. MATERIALS AND METHODSPlasmids, bacterial strains, and growth conditions. The plasmids and the bacterial strains used in this study are listed in Table 1. Escherichia coli DH5␣ was used as a general cloning host. Plasmid DNAs of the pMutin4 derivatives were prepared from E. coli BMH71-18 (recA ϩ ) prior to transformation into B. subtilis. E. coli, and B. subtilis strains were routinely grown in Luria-Bertani broth supplemented with the appropriate antibiotics when necessary (ampicillin at 100 g/ml for E. coli and chloramphenicol at 5 g/ml and erythromycin at 0.3 g/ml for B. subtilis). Standard procedures were used to transform E. coli (15) and B. subtilis (7). Sequencing of PCR-derived DNA fragments in the final plasmid constructs was carried out by Genome Express (Meylan, France).Construction of transcriptional and translational fusions of crh and ptsH to lacZ. To construct the fusion of lacZ to crh-5Ј, the 5Ј region of crh (Ϫ120 to ϩ90) was amplified by using the primers BG9 (crh [Ϫ120 to Ϫ103]) and BG10 (crh [ϩ90 to ϩ73]), digested at the HindIII and BamHI sites within the primers, and inserted between these sites in pMutin4, resulting in plasmid pBGM6. To construct the fusion of lacZ to ptsH-5Ј, the 5Ј region of ptsH (Ϫ276 to ϩ90) was amplified by using the primers LF1 (ptsH [Ϫ276 to Ϫ258]) and LF2 (ptsH [ϩ90 to ϩ73]), digested at the NotI and HindIII sites within the primers, and inserted between these sites in pMutin4, resulting in plasmid pLF2. Plasmid pBGM8 carrying a ⌽(crh-lacZ)(Hyb) fusion gene was constructed by a three-fragment ligation. The entire crh gene with its ribosome binding site (RBS) was amplified by using the primers BG13 (crh [Ϫ20 to Ϫ3]) and BG14 (crh [ϩ255 to ϩ238]) and digested at the HindIII and XhoI sites within the primers. In parallel, the 5Ј part of lacZ was amplified from pMutin4 as a template by using the primers BG15 (pMutin2 [382 to 399]) and BG16 (pMutin2 [698 to 681]) and digested with XhoI (...

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“…The steady-state level of expression of a gene product in growing cells depends both on the promoter activity and the growth rate (31). A fair comparison of expression levels measured at a single time point in terms of promoter activities, as done above, is valid only if the growth rate under the different growth conditions is similar.…”

Section: Resultsmentioning

confidence: 99%

“…A double mutant deficient in both CcpA and RocR showed the highest levels of expression throughout the three growth phases, suggesting that the two repression mechanisms are independent. Expression of citM increased significantly in the stationary growth phase in all strains tested, most likely as a result of the decreased growth rate in this growth phase (31).…”

Section: Discussionmentioning

confidence: 99%

CcpA-Independent Regulation of Expression of the Mg²⁺-Citrate Transporter GenecitMby Arginine Metabolism inBacillus subtilis

2003

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Transcriptional regulation of the Mg2؉ -citrate transporter, CitM, the main citrate uptake system of Bacillus subtilis, was studied during growth in rich medium. Citrate in the growth medium was required for induction under all growth conditions. In Luria-Bertani medium containing citrate, citM expression was completely repressed during the exponential growth phase, marginally expressed in the transition phase, and highly expressed in the stationary growth phase. The repression was relieved when the cells were grown in spent Luria-Bertani medium. The addition of a mixture of 18 amino acids restored repression. L-Arginine in the mixture appeared to be solely responsible for the repression, and ornithine appeared to be an equally potent repressor of citM expression. Studies of mutant strains deficient in RocR and SigL, proteins required for the expression of the enzymes of the arginase pathway, confirmed that uptake into the cell and, most likely, conversion of arginine to ornithine were required for repression. Arginine-mediated repression was independent of a functional CcpA, the global regulator protein in carbon catabolite repression (CCR). Nevertheless, CCR-mediated repression was the major mechanism controlling the expression during exponential growth, while the newly described, CcpA-independent arginine-mediated repression was specifically apparent during the transition phase of growth.

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LacZ-promoter fusions: the effect of growth

Abstract: Microbiology Comment provides a platform for readers of Microbiology to communicate their personal observations and opinions in a more informal way than through the submission of papers.

Cited by 11 publications

References 14 publications

Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+–citrate transporter CitM

Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+–citrate transporter CitM

Drastic Differences in Crh and HPr Synthesis Levels Reflect Their Different Impacts on Catabolite Repression in Bacillus subtilis

CcpA-Independent Regulation of Expression of the Mg²⁺-Citrate Transporter GenecitMby Arginine Metabolism inBacillus subtilis

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LacZ-promoter fusions: the effect of growth

Abstract: Microbiology Comment provides a platform for readers of Microbiology to communicate their personal observations and opinions in a more informal way than through the submission of papers.

Cited by 11 publications

References 14 publications

Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+–citrate transporter CitM

Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+–citrate transporter CitM

Drastic Differences in Crh and HPr Synthesis Levels Reflect Their Different Impacts on Catabolite Repression in Bacillus subtilis

CcpA-Independent Regulation of Expression of the Mg2+-Citrate Transporter GenecitMby Arginine Metabolism inBacillus subtilis

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CcpA-Independent Regulation of Expression of the Mg²⁺-Citrate Transporter GenecitMby Arginine Metabolism inBacillus subtilis