Streptococcus thermophilus strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar. Nevertheless, sequence analysis and complementation studies with Escherichia coli demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes. These were organized into an operon in the order galKTE, which was preceded by a divergently transcribed regulator gene, galR, and followed by a galM gene and the lactose operon lacSZ. Results of Northern blot analysis showed that the structural gal genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the galKTE genes were well expressed in cells grown on lactose or galactose. In both CNRZ 302 and the Gal ؉ mutant NZ302G, the transcription of the galR gene was induced by growth on lactose. Disruption of galR indicated that it functioned as a transcriptional activator of both the gal and lac operons while negatively regulating its own expression. Sequence analysis of the gal promoter regions of NZ302G and nine other independently isolated Gal ؉ mutants of CNRZ 302 revealed mutations at three positions in the galK promoter region, which included substitutions at positions ؊9 and ؊15 as well as a single-base-pair insertion at position ؊37 with respect to the main transcription initiation point. Galactokinase activity measurements and analysis of gusA reporter gene fusions in strains containing the mutated promoters suggested that they were gal promoter-up mutations. We propose that poor expression of the gal genes in the galactose-negative S. thermophilus CNRZ 302 is caused by naturally occurring mutations in the galK promoter.After its discovery almost 40 years ago, the Escherichia coli lactose operon, encoding enzymes of lactose metabolism, became the first model for gene regulation (reviewed in reference 4). The key component of this system is the lac repressor (LacI), the product of the lacI gene. The lac operon contains a primary operator (O 1 ), which is the major element of repression by LacI, and two pseudo-operators, which enhance repressor binding to O 1 by cooperativity. Control of the lac operon also involves activation by the cyclic AMP receptor protein.Many other paradigm systems of negative control have since been described, including GalR, one of the two repressors of the gal regulon encoding enzymes of galactose transport and metabolism in E. coli. Regulation of the gal regulon is mediated through GalR, GalS (Gal isorepressor), and the cyclic AMP receptor protein. GalR and GalS negatively regulate transcription of the two promoters of the gal operon, although GalS is not as efficient as GalR (57).The bioconversion of lactose, which is the primary carbon and energy source in milk, into lactic acid is an essential process in industrial dairy fermentations carried out by lactic acid bacteria. Genetic studies of the metabolic pathways for lactose utilization i...

Streptococcus thermophilus, unlike many other gram-positive bacteria, prefers lactose over glucose as the primary carbon and energy source. Moreover, lactose is not taken up by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) but by the dedicated transporter LacS. In this paper we show that CcpA plays a crucial role in the fine-tuning of lactose transport, ␤-galactosidase (LacZ) activity, and glycolysis to yield optimal glycolytic flux and growth rate. A catabolite-responsive element (cre) was identified in the promoter of the lacSZ operon, indicating a possible role for regulation by CcpA. Transcriptional analysis showed a sevenfold relief of repression in the absence of a functional CcpA when cells were grown on lactose. This CcpA-mediated repression of lacSZ transcription did not occur in wild-type cells during growth on galactose, taken up by the same LacS transport system. Lactose transport during fermentation was increased significantly in strains carrying a disrupted ccpA gene. Moreover, a ccpA disruption strain was found to release substantial amounts of glucose into the medium when grown on lactose. Transcriptional analysis of the ldh gene showed that expression was induced twofold during growth on lactose compared to glucose or galactose, in a CcpA-dependent manner. A reduced rate of glycolysis concomitant with an increased lactose transport rate could explain the observed expulsion of glucose in a ccpA disruption mutant. We propose that CcpA in S. thermophilus acts as a catabolic regulator during growth on the preferred non-PTS sugar lactose. In contrast to other bacteria, S. thermophilus possesses an overcapacity for lactose uptake that is repressed by CcpA to match the rate-limiting glycolytic flux.Carbon catabolite repression (CR) in bacteria is the phenomenon of using a rapidly metabolizable carbon source in the growth medium by inhibiting utilization of other substrates. The mechanism underlying CR is best understood in enteric bacteria, where the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates adenylate cyclase activity. Controlled by the level of cyclic AMP, the cyclic AMP receptor protein is a transcriptional regulator modulating expression of target genes (36, 38). In low-GϩC gram-positive bacteria, the mechanism of CR is distinctly different. The catabolite control protein A (CcpA) is the central regulator of CR, as was shown first for Bacillus subtilis, in which it mediates glucose repression of the ␣-amylase gene (9). CcpA is a member of the LacI-GalR family of bacterial regulator proteins and appears to be widespread among low-GϩC gram-positive bacteria (4,12,21,29). Genes affected by CR typically contain a catabolite-responsive element (cre) near their promoter regions (44). CcpA has been shown to bind to these cre sites in vitro in a way that can be enhanced by indicators of a high energy state in the cell, e.g., glucose 6-phosphate (6, 27). Another important factor in this catabolite control mechanism is the PTS ...

Nisin is a post-translationally modified antimicrobial peptide produced by Lactococcus lactis which binds to lipid II in the membrane to form pores and inhibit cell-wall synthesis. A nisinresistant (Nis R ) strain of L. lactis, which is able to grow at a 75-fold higher nisin concentration than its parent strain, was investigated with respect to changes in the cell wall. Direct binding studies demonstrated that less nisin was able to bind to lipid II in the membranes of L. lactis Nis R than in the parent strain. In contrast to vancomycin binding, which showed ring-like binding, nisin was observed to bind in patches close to cell-division sites in both the wild-type and the Nis R strains. Comparison of modifications in lipoteichoic acid of the L. lactis strains revealed an increase in D-alanyl esters and galactose as substituents in L. lactis Nis R , resulting in a less negatively charged cell wall. Moreover, the cell wall displays significantly increased thickness at the septum. These results indicate that shielding the membrane and thus the lipid II molecule, thereby decreasing abduction of lipid II and subsequent pore-formation, is a major defence mechanism of L. lactis against nisin.

The adaptation to utilise lactose as primary carbon and energy source is a characteristic for Streptococcus thermophilus. These organisms, however only utilise the glucose moiety of lactose while the galactose moiety is excreted into the growth medium. In this study we evaluated the diversity of sugar utilisation and the conservation of the gal-lac gene cluster in a collection of 18 S. thermophilus strains isolated from a variety of sources. For this purpose analysis was performed on DNA from these isolates and the results were compared with those obtained with a strain from which the complete genome sequence has been determined. The sequence, organisation and flanking regions of the S. thermophilus gal-lac gene cluster were found to be highly conserved among all strains. The vast majority of the S. thermophilus strains were able to utilize only glucose, lactose, and sucrose as carbon sources, some strains could also utilize fructose and two of these were able to grow on galactose. Molecular characterisation of these naturally occurring Gal+ strains revealed up-mutations in the galKTE promoter that were absent in all other strains. These data support the hypothesis that the loss of the ability to ferment galactose can be attributed to the low activity of the galKTE promoter, probably as a consequence of the adaptation to milk in which the lactose levels are in excess.

Before leaving the site of transcription, newborn messenger RNAs (mRNAs) become associated with a number of different proteins. How these large messenger ribonucleoprotein (mRNP) complexes then move through the dense nucleoplasm to reach the nuclear periphery has been a fascinating question for the last few years. We have studied the mechanism of this process by tracking individual mRNPs in real time. We were able to track mRNPs at single-molecule resolution because we utilized mRNAs that were engineered to have a sequence motif repeated 96 times in their untranslated region. These mRNAs were visualized with the help of molecular beacons that were specific for the repeated sequence; the binding of 96 molecular beacons to each mRNA molecule rendered them so intensely fluorescent that they were visible as fine fluorescent spots that could be tracked by high-speed video microscopy. In this chapter, we describe the details of the construction of genes containing the tandem repeats, the integration of such genes into the genome of a cell line, the design and testing of molecular beacons, time-lapse imaging of mRNPs, and computer-aided generation and analysis of the tracks of the individual mRNPs. These methods will be useful for studies of other dynamic processes such as mRNA export, splicing, and decay.