In vivo effect of mutations in the antiterminator LacT in Lactobacillus casei

Gosalbes, María José; Esteban, Carlos David; Pérez-Martı́nez, Gaspar

doi:10.1099/00221287-148-3-695

Cited by 18 publications

(29 citation statements)

References 54 publications

(9 reference statements)

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“…On the other hand, our Northern analysis showed that the transcription of the lacTEGF operon of strain R1 could be induced by lactose but suppressed by glucose. This in fact was consistent with the dual regulation mechanism reported for L. casei (Alpert & Siebers, 1997;Gosalbes et al, 1997Gosalbes et al, , 1999Gosalbes et al, , 2002Monedero et al, 1997). The inducible, catabolite-repressed expression of the lacTEGF operon detected here suggests that L. casei ATCC 27139 (Lac 2 ) originally might have been Lac + , but was converted to Lac 2 .…”

Section: Discussionsupporting

confidence: 80%

“…L. casei strains were cultured at 37 uC in MannRogosa-Sharpe (MRS; Difco) broth or Lactobacillus-carrying medium (LCM) supplemented with 0.5 or 1 % filter-sterilized carbohydrates (Efthymiou & Hansen, 1962). Ribose (non-inducing, non-repressing sugar), lactose (inducer) and glucose (repressor) were used as carbohydrates as previously described for studying the regulation of the lacTEGF operon in L. casei (Alpert & Siebers, 1997;Gosalbes et al, 1997Gosalbes et al, , 1999Gosalbes et al, , 2002Monedero et al, 1997). For preparing the agar plates, 1.5 % agar (Amresco) was added to the medium.…”

Section: Methodsmentioning

confidence: 99%

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Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac− to Lac+

Tsai

Chen

et al. 2009

Microbiology

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Lactose metabolism is a changeable phenotype in strains of Lactobacillus casei. In this study, we found that L. casei ATCC 27139 was unable to utilize lactose. However, when exposed to lactose as the sole carbon source, spontaneous Lac + clones could be obtained. A gene cluster (lacTEGF-galKETRM) involved in the metabolism of lactose and galactose in L. casei ATCC 27139 (Lac " ) and its Lac + revertant (designated strain R1) was sequenced and characterized. We found that only one nucleotide, located in the lacTEGF promoter (lacTp), of the two lac-gal gene clusters was different. The protein sequence identity between the lac-gal gene cluster and those reported previously for some L. casei (Lac + ) strains was high; namely, 96-100 % identity was found and no premature stop codon was identified. A single point mutation located within the lacTp promoter region was also detected for each of the 41 other independently isolated Lac + revertants of L. casei ATCC 27139. The revertants could be divided into six classes based on the positions of the point mutations detected. Primer extension experiments conducted on transcription from lacTp revealed that the lacTp promoter of these six classes of Lac + revertants was functional, while that of L. casei ATCC 27139 was not. Northern blotting experiments further confirmed that the lacTEGF operon of strain R1 was induced by lactose but suppressed by glucose, whereas no blotting signal was ever detected for L. casei ATCC 27139. These results suggest that a single point mutation in the lacTp promoter was able to restore the transcription of a fully functional lacTEGF operon and cause a phenotype switch from Lac " to Lac + for L. casei ATCC 27139.

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Section: Discussionsupporting

confidence: 80%

Section: Methodsmentioning

confidence: 99%

Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac− to Lac+

Tsai

Chen

et al. 2009

Microbiology

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“…Exchange of this histidine leads to constitutively high antitermination activity in vivo even in the absence of the specific substrate (20,24,25). The second histidine in PRD1 participates in negative control of LicT and LacT (20,25). In contrast, the histidine(s) in PRD2 were shown to be necessary for activity of the dually controlled proteins SacT, LicT, and LacT (6,20,25), and preferential phosphorylation of these residues by HPr has been demonstrated for LicT (19).…”

mentioning

confidence: 98%

“…Where investigated, the first conserved histidine in PRD1 was found to be crucial for negative control. Exchange of this histidine leads to constitutively high antitermination activity in vivo even in the absence of the specific substrate (20,24,25). The second histidine in PRD1 participates in negative control of LicT and LacT (20,25).…”

mentioning

confidence: 99%

“…The second histidine in PRD1 participates in negative control of LicT and LacT (20,25). In contrast, the histidine(s) in PRD2 were shown to be necessary for activity of the dually controlled proteins SacT, LicT, and LacT (6,20,25), and preferential phosphorylation of these residues by HPr has been demonstrated for LicT (19). Based on these observations, it has been proposed that these proteins are stimulated by HPr-dependent phosphorylation of the histidines in PRD2 and inhibited by EII-dependent phosphorylation of the first histidine in PRD1.…”

mentioning

confidence: 99%

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Regulation of the Escherichia coli Antiterminator Protein BglG by Phosphorylation at Multiple Sites and Evidence for Transfer of Phosphoryl Groups between Monomers

Görke

2003

Journal of Biological Chemistry

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Activity of antiterminator protein BglG regulating the ␤-glucoside operon in Escherichia coli is controlled by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in a dual manner. It requires HPr phosphorylation to be active, whereas phosphorylation by the ␤-glucoside-specific transport protein EII Bgl inhibits its activity. BglG and its relatives carry two PTS regulation domains (PRD1 and PRD2), each containing two conserved histidines. For BglG, histidine 208 in PRD2 was reported to be the negative phosphorylation site. In contrast, other antiterminators of this family are negatively regulated by phosphorylation of the first histidine in PRD1, and presumably activated by phosphorylation of the histidines in PRD2. In this work, a screen for mutant BglG proteins that escape repression by EII Bgl yielded exchanges of nine residues within PRD1, including conserved histidines His-101 and His-160, and C-terminally truncated proteins. Genetic and phosphorylation analyses indicate that His-101 in PRD1 is phosphorylated by EII Bgl and that His-160 contributes to negative regulation. His-208 in PRD2 is essential for BglG activity, suggesting that it is phosphorylated by HPr. Surprisingly, phosphorylation by HPr is not fully abolished by exchanges of His-208. However, phosphorylation by HPr is inhibited by exchanges in PRD1 and the phosphorylation of these mutants is restored in the presence of wild-type BglG. These results suggest that the activating phosphoryl group is transiently donated from HPr to PRD1 and subsequently transferred to His-208 of a second BglG monomer. The active His-208-phosphorylated BglG dimer can subsequently be inhibited in its activity by EII Bgl -catalyzed phosphorylation at His-101.

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