The lac operon of Lactobacillus casei contains lacT, a gene coding for a protein of the Bg1G family of transcriptional antiterminators

Abstract

The 5 region of the lac operon of Lactobacillus casei has been investigated. An open reading frame of 293 codons, designated lacT, was identified upstream of lacE. The gene product encoded by lacT is related to the family of transcriptional antiterminator proteins, which includes BglG from Escherichia coli, ArbG from Erwinia chrysanthemi, SacT, SacY, and LicT from Bacillus subtilis, and BglR from Lactococcus lactis. Amino acid sequence identities range from 35 to 24%, while similarities range from 56 to 47%. The transcriptional start site of the lac operon was identified upstream of lacT. The corresponding mRNA would contain in the 5 region a sequence with high similarity to the consensus RNA binding site of transcriptional antiterminators overlapping a sequence capable of folding into a structure that resembles a rho-independent terminator. LacT was shown to be active as an antiterminator in a B. subtilis test system using the sacB target sequence. lacT directly precedes lacEGF, the genes coding for enzyme IICB, phospho-␤-galactosidase, and enzyme IIA, and these genes are followed by a sequence that appears to encode a second rho-independent transcription terminator-like structure. Northern hybridizations with probes against lacT, lacE, and lacF revealed transcripts of similar sizes for the lac mRNAs of several L. casei strains. Since the length of the lac mRNA is just sufficient to contain lacTEGF, we conclude that the lac operon of L. casei does not contain the genes of the accessory tagatose-6-phosphate pathway as occurs in the lac operons of Lactococcus lactis, Streptococcus mutans, or Staphylococcus aureus.Uptake of lactose into bacterial cells and initiation of its metabolism can be mediated by several pathways: ABC protein-dependent systems, lactose-galactose antiporters, lactose-H ϩ symport systems, or the lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) (20). While ABC protein-dependent lactose transport has been demonstrated in Agrobacterium radiobacter (59), lactose-galactose antiport has been described for Streptococcus thermophilus (41). The lactose permease-H ϩ symport system, the genetics of which represent the paradigm of bacterial operon organization and regulation (31), is found in the enterobacteria, and its function has been studied in Lactococcus lactis (34). Lactose permease-␤-galactosidase systems have also been identified in other gram-positive species, including lactobacilli (23, 32). The alternative pathway for lactose uptake, however, the lactosespecific PTS (Lac-PTS), has so far been confirmed only in gram-positive bacteria such as Staphylococcus aureus, Streptococcus mutans, dairy lactococci, and Lactobacillus casei strains (2,3,19,20,29,37,40,42,46). In some Lactobacillus species, two pathways, Lactose permease-␤-galactosidase and Lac-PTS, have been found to coexist (23,44).During PTS-mediated transport, lactose is phosphorylated and then hydrolyzed by phospho-␤-galactosidase (P-␤-Gal) to glucose and galactose-6-P. While glucose is channeled into the E...

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

“…Furthermore, the transcript accumulation of the lacTEGF operon in L. casei ATCC 27139 (Lac 2 ) and that of the corresponding Lac + revertant (strain R1) was studied by Northern blot analysis. A complete lacTEGF gene transcript in strain R1 was detected, similar to those observed by others for other L. casei (Lac + ) strains (Alpert & Siebers, 1997). However, no blotting signal was detected for L. casei ATCC 27139 (Lac 2 ).…”

“…2b). We found that, except for the promoter region, the sequences of these aforementioned regulatory regions were exactly the same as those reported for the Lac + strains L. casei BL23, 64H and ATCC 334 (Alpert & Siebers, 1997;Gosalbes et al, 1997;Makarova et al, 2006) (Supplementary Fig. S2).…”

“…The distance between the 210 regions and the transcriptional start sites determined was 6 nt for strains R1, R27, R37, R39 and R42, and 5 nt for strain R41, all of which fall within the range of 6.9±2.7 bp reported for some lactobacilli (McCracken et al, 2000). Moreover, the detected transcriptional start site coincided with that of L. casei 64H (Lac + ) (Alpert & Siebers, 1997). However, no such signal was detected for L. casei ATCC 27139 (Lac 2 ) grown in LCM supplemented with 0.5 % glucose, 0.5 % ribose, 0.5 % ribose plus 0.5 % lactose, or 0.5 % glucose plus 0.5 % lactose.…”

“…These Lac + revertants were divided into six classes based on the detected mutation. For class I revertants, a C-to-T substitution in the 210 box region caused a change of the original Lac 2 promoter sequence from TTTACA-N16-TACAAC to TTTACA-N16-TACAAT, and the lacTp promoter sequence was found to be identical to that of the functional lacTp promoter of L. casei 64H (Alpert & Siebers, 1997). In class II and III revertants, an insertion of one base had occurred in the region between the 235 and the 210 boxes of the lacTp promoter.…”

“…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.…”