The EIIABMan phosphotransferase system permease regulates carbohydrate catabolite repression in Streptococcus gordonii

Commensal oral streptococci play critical roles in oral biofilm formation and promote dental health by competing with, and antagonizing the growth of, pathogenic organisms, such as Streptococcus mutans. Efficient utilization of the spectrum of carbohydrates in the oral cavity by commensal streptococci is essential for their persistence, and yet very little is known about the regulation of carbohydrate catabolism by these organisms. Carbohydrate catabolite repression (CCR) in the abundant oral commensal Streptococcus gordonii strain DL-1 was investigated using the exo-β-D-fructosidase gene (fruA) and a fructose/mannose sugar:phosphotransferase (PTS) enzyme II operon (levDEFG) as model systems. Functional studies confirmed the predicted roles of FruA and LevD in S. gordonii. ManL, the AB domain of a fructose/mannose-type enzyme II PTS permease, contributed to utilization of glucose, mannose, galactose, and fructose and exerted primary control over CCR of the fruA and levD operons. Unlike in S. mutans, ManL-dependent CCR was not sugar specific, and galactose was very effective at eliciting CCR in S. gordonii. Inactivation of the apparent ccpA homologue of S. gordonii actually enhanced CCR of fruA and levD, an effect likely due to its demonstrated role in repression of manL expression. Thus, there are some similarities and fundamental differences in CCR control mechanisms between the oral pathogen S. mutans and the oral commensal S. gordonii that may eventually be exploited to enhance the competitiveness of health-associated commensals in oral biofilms.     

Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon

The levanase gene (sacC) of Bacillus subtilis is the distal gene of a fructose-inducible operon containing five genes. The complete nucleotide sequence of this operon was determined. The first four genes levD, levE, levF and levG encode polypeptides that are similar to proteins of the mannose phosphotransferase system of Escherichia coli. The levD and levE gene products are homologous to the N and C-terminal part of the enzyme IIIMan, respectively, whereas the levF and levG gene products have similarities with the enzymes IIMan. Surprisingly, the polypeptides encoded by the levD, levE, levF and levG genes are not involved in mannose uptake, but form a fructose phosphotransferase system in B. subtilis. This transport is dependent on the enzyme I of the phosphotransferase system (PTS) and is abolished by deletion of levF or levG and by mutations in either levD or levE. Four regulatory mutations (sacL) leading to constitutive expression of the lavanase operon were mapped using recombination experiments. Three of them were characterized at the molecular level and were located within levD and levE. The levD and levE gene products that form part of a fructose uptake PTS act as negative regulators of the operon. These two gene products may be involved in a PTS-mediated phosphorylation of a regulator, as in the bgl operon of E. coli.     

Fructose transport in Bacillus subtilis.

The transport of fructose in Bacillus subtilis was studied in various mutant strains lacking the following activities: ATP-dependent fructokinase (fruC), the fructose 1-phosphate kinase (fruB) the phosphofructokinase (pfk), the enzyme I of the phosphoenolpyruvate phosphotransferase system (the thermosensitive mutation ptsI1), and a transport activity (fruA). Combinations of these mutations indicated that the transport of fructose in Bacillus subtilis is tightly coupled to its phosphorylation either in fructose 1-phosphate, identified in vivo and in vitro or in fructose 6-phosphate identified by indirect lines of evidence. These steps of fructose metabolism were shown to depend on the activity of the enzyme I of the phosphoenolpyruvate phosphotransferase systems. The fruA mutations affect the transport of fructose when the bacteria are submitted to catabolite repression. The mutations were localized on the chromosome of Bacillus subtilis in a cluster including the fruB gene. When grown in a medium supplemented by a mixture of potassium glutamate and succinate the fruA mutants are able to carry on the two vectorial metabolisms generating fructose 6-phosphate as well as fructose 1-phosphate. A negative search of strictly negative transport mutants in fruA strains indicated that more than two structural genes are involved in the transport of fructose.     

The multifunctional fructose-specific component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli K12--fruA gene product

Mutational damage of the ptsH gene leads to pleiotropic disturbance of sugar utilization in Escherichia coli K12. A fruS mutation suppresses the defect because of a constitutional expression of the fruB and fruA genes. FruB protein possessing a pseudo-HPr activity replaces the HPr. It was shown that wild type allele fruS+ dominates over the fruS1156 mutation in heterozygous merodiploid. The existence of thermosensitive mutations (fruS4 and fruS12) which repair the ptsH damage was also demonstrated. The fruS mutations were located in the fru operon. Fructose utilization was not disturbed in fruS1156 mutant, but fruS2 and fruS12 mutants were unable to utilize fructose. Spontaneous mutations (fruS6 and fruS13) possessing the same phenotype at any temperature similar to the thermosensitive ones under nonpermissive conditions were isolated. They were mapped using the P1vir transduction. The fruS mutations were found in the structural gene of the fructose operon. Presumably it is the fruA gene that cods for the fructose-specific multidomain protein IIB'Bc of the phosphoenolpyruvate-dependent phosphotransferase system.     

Fructose catabolism in Xanthomonas campestris pv. campestris. Sequence of the PTS operon, characterization of the fructose-specific enzymes.

In Xanthomonas campestris pv. campestris, fructose is transported and phosphorylated into fructose 1-phosphate through a phosphoenolpyruvate-dependent phosphotransferase system. The nucleotide sequence of the fruA gene encoding the phosphotransferase system permease specific of fructose (EIIFru) was determined. The fructose 1-phosphate produced by the phosphotransferase system is phosphorylated into fructose 1,6-bisphosphate by a 1-phosphofructokinase. This enzyme was characterized and the corresponding gene (fruK) was sequenced. Sequence comparisons revealed that FruK is a member of a new family of ATP-binding proteins composed of sugar (or sugar-phosphate) kinases. In phosphotransferase system-deficient strains, fructose can still be transported by an unidentified permease. The intracellular fructose is then phosphorylated by a multimeric fructokinase of 135 kDa specific for fructose and inhibited by fructose, fructose 1,6-bisphosphate, and mannose. Several other enzymes of fructose metabolism were assayed and a potential pathway for fructose catabolism is presented.     

Routes for fructose utilization by Escherichia coli

There are three main routes for the utilization of fructose by Escherichia coli. One (Route A) predominates in the growth of wild-type strains. It involves the functioning of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and a fructose operon, mapping at min. 48.7, containing genes for a membrane-spanning protein (fruA), a 1-phosphofructose kinase (fruK) and a diphosphoryl transfer protein (fruB), under negative regulation by a fruR gene mapping at min. 1.9. A second route (Route B) also involves the PTS and membrane-spanning proteins that recognize a variety of sugars possessing the 3,4,5-D-arabino-hexoseconfiguration but with primary specificity for mannose(manXYZ), mannitol (mtlA) and glucitol (gutA) and which, if over-produced, can transport also fructose. A third route (Route C), functioning in mutants devoid of Routes A and B, does not involve the PTS: fructose diffuses into the cell via an isoform (PtsG-F) of the major glucose permease of the PTS and is then phosphorylated by ATP and a manno(fructo)kinase (Mak+) specified by a normally cryptic 1032 bp ORF (yajF) of hitherto unknown function (Mak-o), mapping at min. 8.8 and corresponding to a peptide of 344 amino acids. Conversion of the Mak-o to the Mak+ phenotypeinvolves an A24D mutation in a putative regulatory region.     

Characterization of Streptococcus mutans strains deficient in EIIAB Man of the sugar phosphotransferase system

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIAB(Man) (encoded by manL) in gene regulation and sugar transport was investigated in Streptococcus mutans UA159. The manL knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIAB(Man) is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (fruI) and constitutive (fruCD) EII fructose, revealed that S. mutans EIIAB(Man) transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIAB(Man) controlled expression of the known virulence genes, glucosyltransferases (gtfBC) and fructosyltransferase (ftf) promoter fusions of these genes were established in the wild-type and EIIAB(Man)-deficient strains. In the manL mutant, the level of chloramphenicol acetyltransferase activity expressed from the gtfBC promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIAB(Man) is required for optimal expression of gtfBC. No significant differences were observed between the mutant and the wild-type background in ftf regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in ftf expression. Two-dimensional gel analysis of batch-grown cells of the EIIAB(Man)-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIAB(Man) has a pleiotropic effect on gene expression.     

Uncoupling expression of genes coding for the synthesis of proteins involved in transport and utilization of fructose in Escherichia coli K-12]

A novel mutation fruS localised in the fru operon has been obtained. The mutation uncouples expression of genes determining fructose specific uptake and utilization. In the fruS bacteria fruA and fruF genes (coding for enzyme II and FPr, respectively) become constitutive, while the fruK gene (responsible for fructose-1-phosphate kinase synthesis) remains inducible. In contrast to the already known mutations making the whole fru operon constitutive, the fruS mutation: 1) does not lead to xylitol sensitivity; 2) does not depress growth on lactate, pyruvate and alanine; 3) does not decrease PEP-synthase activity.     

Analysis of dofA,a fruA-dependent developmental gene,and its homologue,dofB, in Myxococcus xanthus

The developmentally regulated gene dofA, identified from pulse-labeling experiments by two-dimensional gel electrophoresis, and its homologue, dofB, were cloned and characterized in Myxococcus xanthus. Deletion of dofA and dofB did not affect the vegetative growth and development of M. xanthus. dofA was specifically expressed during development, while dofB expression was observed during vegetative growth and development. The dofA-lacZ fusion was introduced into a fruA mutant and A, B, C, D, and E extracellular signal mutants. The pattern of dofA expression in the C signal mutant was similar to that of the wild-type strain, while dofA expression was not detected in the fruA mutant. These results are consistent with those of the pulse-labeling experiments. dofA expression was reduced in A and E signal mutants, whereas dofA expression was delayed in B and D signal mutants. The patterns of expression of the dofA gene in the fruA mutant and the five signal mutants are strikingly similar to that of the tps gene, which encodes protein S, a major component of the outer surface of the myxospore; this result suggests that the dofA and tps genes are similarly regulated. The involvement of a highly GC-rich inverted repeat sequence (underlined), CGGCCCCCGATTCGTCGGGGGCCG, in developmentally regulated dofA expression is suggested.     

黄色黏细菌转录因子FruA对自身基因的表达不具自调节作用

 粘细菌是研究多细胞结构形态发生机制的良好模型.FruA是粘细菌发育所必需的一种 关键性转录因子, 调节一系列发育相关基因的表达,本文研究FruA对自身基因是否存在反馈调节从而导致发育后期fruA表达水平的下调.以野生型粘细菌模式菌株DK1622为基础构建fruA基因敲除突变株DK1622ΔfruA,再将fruA-lacZ转录融合载体pMF1A整合入fruA突变株染色体attB, 获得重组菌株DK1622ΔfruA/pMF1A,通过检测β-半乳糖苷酶活性来确认FruA对自身基因的表达水平是否有影响. 结果表明fruA调控序列完整的fruA-lacZ转录融合体β-半乳糖苷酶活性在DK1622/pMF1A和DK1622ΔfruA/pMF1A之间无明显差异, 即fruA表达产物作为一种转录因子对自身基因的转录没有调节作用,黏细菌发育后期fruA表达水平的下降存在其它调节机制.