Utilization of D-ribitol by Lactobacillus casei BL23 requires a mannose-type phosphotransferase system and three catabolic enzymes.

Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment D-ribitol (also called D-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates D-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in D-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented D-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a D-ribitol-5-phosphate (D-ribitol-5-P) 2-dehydrogenase, a D-ribulose-5-P 3-epimerase, a D-ribose-5-P isomerase, and a D-xylulose-5-P phosphoketolase. In the first catabolic step, the protein D-ribitol-5-P 2-dehydrogenase uses NAD(+) to oxidize D-ribitol-5-P formed during PTS-catalyzed transport to D-ribulose-5-P, which, in turn, is converted to D-xylulose-5-P by the enzyme D-ribulose-5-P 3-epimerase. Finally, the resulting D-xylulose-5-P is split by D-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate D-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as D-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation.

Role of α-phosphoglucomutase and phosphoglucose isomerase activities at the branching point between sugar catabolism and anabolism in Lactobacillus casei.

AIMS: To evaluate the role of α-phosphoglucomutase (α-Pgm) and phosphoglucose isomerase (Pgi) activities in growth rate, sugar-phosphates, UDP-sugars and lactate biosynthesis in Lactobacillus casei. METHODS AND RESULTS: The pgm and pgi genes coding for α-Pgm and Pgi activities in L. casei BL23, respectively, were identified, cloned and shown to be functional by homologous overexpression. In MRS fermentation medium with glucose, overexpression of pgm gene in L. casei resulted in a growth rate reduced to 75% and glucose-6P levels reduced to 47%. By contrast, with lactose, the growth rate was raised to 119%. An increment of α-Pgm activity had no significant effect on UDP-sugar levels. Remarkably, Pgi overexpression in L. casei grown in lactose or galactose resulted in almost a double growth rate with respect to the control strain. The increased Pgi activity also resulted in glucose-6P levels reduced to 25 and 59% of control strain cultured in glucose and lactose, respectively, and the fructose-6P levels were increased to 128% on glucose. UDP-glucose and UDP-galactose levels were reduced to 66 and 55%, respectively, of control strain levels cultured in galactose. In addition, the lactate yield increased to 115% in the strain overproducing Pgi grown in galactose. CONCLUSIONS: The physiological amount of α-Pgm and Pgi activities is limited for L. casei growth on lactose, and lactose and galactose, respectively, and that limitation was overcome by pgm and pgi gene overexpression. The increment of α-Pgm and Pgi activities, respectively, resulted in modified levels of sugar-phosphates, sugar-nucleotides and lactate showing the modulation capacity of the carbon fluxes in L. casei at the level of the glycolytic intermediate glucose-6P. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the role of key enzymes in metabolic fluxes at the branching point between anabolic and catabolic pathways would allow a rational design of engineering strategies in L. casei.

Influence of the carbohydrate source on beta-glucan production and enzyme activities involved in sugar metabolism in Pediococcus parvulus 2.6.

The influence of carbohydrate source on growth, exopolysaccharide (EPS) production and on the activity of the enzymes implicated in energy generation and UDP-glucose synthesis in Pediococcus parvulus 2.6 was evaluated. The highest EPS production was obtained on glucose, while fructose was a poor substrate for EPS synthesis. HPLC and NMR analysis on monomer composition and structure of the EPS showed that this strain produced the same beta-glucan, regardless of the carbohydrate source. The alpha-phosphoglucomutase specific activities were dependent on the carbohydrate source and a high correlation between the activity of this enzyme and the amount of EPS was found in glucose- and maltose-grown cultures. alpha-UDP-glucose pyrophosphorylase activity, necessary for the activation of glucose, was very low, but significantly higher on glucose as sugar source. In vitro phosphorylation assays and transport activities showed that glucose is taken up by a proton motive force-dependent permease, while fructose is internalized by an inducible phosphotransferase system, which renders fructose-6-phosphate. The levels of 6-phosphofructokinase activity and alpha-phosphoglucomutase activities determined on fructose were higher and lower, than those found on glucose or maltose, respectively. This suggests that fructose-6-phosphate is mainly diverted to glycolysis and explains the low EPS synthesis on fructose. Results indicate that alpha-phosphoglucomutase and/or alpha-UDP-glucose pyrophosphorylase might be the bottlenecks for EPS biosynthesis, opening the field for metabolic-engineering strategies aimed to improve EPS production.

Genetics of L-sorbose transport and metabolism in Lactobacillus casei.

Genes encoding L-sorbose metabolism of Lactobacillus casei ATCC 393 have been identified on a 6.8-kb chromosomal DNA fragment. Sequence analysis revealed seven complete genes and a partial open reading frame transcribed as two units. The deduced amino acid sequences of the first transcriptional unit (sorRE) showed high similarity to the transcriptional regulator and the L-sorbose-1-phosphate reductase of the sorbose (sor) operon from Klebsiella pneumoniae. The other genes are transcribed as one unit (sorFABCDG) in opposite direction to sorRE. The deduced peptide sequence of sorF showed homology with the D-sorbitol-6-phosphate dehydrogenase encoded in the sor operon from K. pneumoniae and sorABCD to components of the mannose phosphotransferase system (PTS) family but especially to domains EIIA, EIIB, EIIC and EIID of the phosphoenolpyruvate-dependent L-sorbose PTS from K. pneumoniae. Finally, the deduced amino acid sequence of a truncated gene (sorG) located downstream of sorD presented high similarity with ketose-1,6-bisphosphate aldolases. Results of studies on enzyme activities and transcriptional analysis revealed that the two gene clusters, sorRE and sorFABCDG, are induced by L-sorbose and subject to catabolite repression by D-glucose. Data indicating that the catabolite repression is mediated by components of the PTS elements and by CcpA, are presented. Results of sugar uptake assays in L. casei wild-type and sorBC mutant strains indicated that L-sorbose is taken up by L-sorbose-specific enzyme II and that L. casei contains an inducible D-fructose-specific PTS. Results of growth analysis of those strains and a man sorBC double mutant suggested that L-sorbose is probably also transported by the D-mannose PTS. We also present evidence, from studies on a sorR mutant, suggesting that the sorR gene encodes a positive regulator of the two sor operons. Sequence alignment of SorR, SorC (K. pneumoniae), and DeoR (Bacillus subtilis) revealed that they might constitute a new group of transcriptional regulators.

Expression and secretion of Bacillus polymyxa neopullulanase in Saccharomyces cerevisiae.

We have isolated the gene encoding the neopullulanase enzyme from Bacillus polymyxa CECT 155. It consists of an open reading frame of 1545 bp that could code for a protein of 515 amino acids. This open reading frame was expressed in Bacillus subtilis and the corresponding transformants produced extracellular neopullulanase. The neopullulanase gene was also expressed in Saccharomyces cerevisiae placing it under the control of the yeast actin gene (ACT1) promoter. Clones containing the intact neopullulanase gene, including its own bacterial signal sequence, gave rise to the synthesis of active, but intracellular, enzyme by S. cerevisiae transformants. When sequences specifying the signal sequence and leader region of the yeast mating pheromone alpha-factor (MF alpha 1) were fused upstream of the gene encoding the neopullulanase enzyme, the enzyme was secreted by S. cerevisiae. The secreted protein presented the same biochemical properties and the same apparent molecular mass as the Bacillus polymyxa original enzyme. The predicted amino acid sequence of the neopullulanase protein contained sequence motifs conserved among amylolytic enzymes. Northern blot analysis indicated that the transcription of the neopullulanase gene in B. polymyxa was induced by the presence of the substrate, pullulan, in the culture, and was repressed by glucose.

Lack of correlation between binding of EcoRII methyltransferase to DNA duplexes containing mismatches and the promotion of C to T mutations.

The cytosine methyltransferases (MTases) M. HhaI and M. HpaII bind substrates in which the target cytosine is replaced by uracil or thymine, i.e. DNA containing a U:G or a T:G mismatch. We have extended this observation to the EcoRII MTase (M. EcoRII) and determined the apparent Kd for binding. Using a genetic assay we have also tested the possibility that MTase binding to U:G mismatches may interfere with repair of the mismatches and promote C:G to T:A mutations. We have compared two mutants of M. EcoRII that are defective for catalysis by the wild-type enzyme for their ability to bind DNA containing U:G or T:G mismatches and for their ability to promote C to T mutations. We find that although all three proteins are able to bind DNAs with mismatches, only the wild-type enzyme promotes C:G to T:A mutations in vivo. Therefore, the ability of M. EcoRII to bind U:G mismatched duplexes is not sufficient for their mutagenic action in cells.

A cytosine methyltransferase converts 5-methylcytosine in DNA to thymine.

Sites of cytosine methylation are known to be hot spots for C.G to T.A mutations in a number of systems, including human cells. Traditionally, spontaneous hydrolytic deamination of 5-methylcytosine to thymine has been invoked as the cause of this phenomenon. We show here that a bacterial cytosine methyltransferase can convert 5-methylcytosine in DNA to thymine and that this reaction creates a mutational hot spot at a site of DNA methylation. The reaction is fairly insensitive to the methyl donor in the reaction, S-adenosylmethionine. In many cancers, the most frequent class of mutations is C to T changes within CG dinucleotides of the tumor suppressor gene p53. Because of the similarities of the reaction mechanisms of mammalian and bacterial enzymes and the physiology of the cancer cells, this reaction is expected to contribute to mutations at CG dinucleotides in precancerous cells.

A novel repetitive sequence lies near the gene encoding a cytosine methyltransferase in the cyanobacterium Dactylococcopsis salina.

An unusual cluster of tandemly repeated DNA sequences (TRS) was found downstream from the gene encoding DsaV methyltransferase, the DNA modification enzyme in the DsaV restriction-modification system found in a strain of Dactylococcopsis salina (Ds). The repeat unit is about 32-bp long and is present 13 times in the cluster. Each repeat unit can be divided into two distinct parts based on the level of sequence conservation and evolution. Hybridization of Ds DNA with a probe specific for this cluster revealed that there were at least two additional sites within the genome with similar TRS. The TRS units are localized in one region of the Ds genome. They do not share significant sequence similarity with other TRS found in prokaryotes.

Cloning and characterization of the gene encoding the DsaV methyltransferase.

A gene encoding the M.DsaV methyltransferase was cloned and characterized. The enzyme methylates the internal cytosines in the 5'-CCTGG recognition sequence, as determined by a novel rapid method employing 3H label and exonuclease III.

Effect of 5-azacytidine and sinefungin on Streptomyces development.

The effect of two DNA-methyltransferase inhibitors, 5-azacytidine (5azaC) and sinefungin (Sf), on the development of Streptomyces antibioticus ETH7451 (Sa) was studied. Pulse labeling experiments and SDS-PAGE analysis of proteins from cells grown in sporulation synthetic medium showed that both inhibitors affect a limited number of systems. Synthesis of the antibiotic rhodomycin was increased in the presence of 5azaC. 5azaC also stimulated the production of actinorhodin in cultures of S. coelicolor A3(2) grown in minimal medium. The analog did not affect the expression of whiB and whiG, two sporulation genes from S. coelicolor A3(2) whose homologues are present in Sa. Overall results indicated that 5azaC and Sf affect specific events associated with differentiation and secondary metabolism in Streptomyces.

DsaV methyltransferase and its isoschizomers contain a conserved segment that is similar to the segment in Hhai methyltransferase that is in contact with DNA bases.

The methyltransferase (MTase) in the DsaV restriction--modification system methylates within 5'-CCNGG sequences. We have cloned the gene for this MTase and determined its sequence. The predicted sequence of the MTase protein contains sequence motifs conserved among all cytosine-5 MTases and is most similar to other MTases that methylate CCNGG sequences, namely M.ScrFI and M.SsoII. All three MTases methylate the internal cytosine within their recognition sequence. The 'variable' region within the three enzymes that methylate CCNGG can be aligned with the sequences of two enzymes that methylate CCWGG sequences. Remarkably, two segments within this region contain significant similarity with the region of M.HhaI that is known to contact DNA bases. These alignments suggest that many cytosine-5 MTases are likely to interact with DNA using a similar structural framework.

The effect of sinefungin and synthetic analogues on RNA and DNA methyltransferases from Streptomyces.

Sinefungin is an antibiotic structurally related to S-adenosylmethionine. It has been described as an inhibitor of RNA transmethylation reactions in viruses and eukaryotic organisms, but not in bacteria. We show here that sinefungin strongly inhibits RNA methyltransferase activity, but not the biosynthesis of these enzymes in Streptomyces. All the methylated bases found in Streptomyces RNA (1-methyladenine, N6-methyladenine, N6,N6-dimethyladenine and 7-methylguanine) are inhibited by this antibiotic. Experiments with sinefungin analogues show that specific changes in the ornithine radical of the molecule still preserve its inhibitory capability. The substitution of the adenine radical by uridine causes the loss of the inhibitory effect. These results and our former studies on Streptomyces DNA methylation, suggest that nucleic acid modification is the main target of sinefungin in Streptomyces.

Characterization of Rrh4273I, a restriction-modification system of Rhodococcus rhodochrous ATCC 4273 (Nocardia corallina) which recognizes the same sequence as the Streptomyces albus G SalI restriction-modification system.

Rhodococcus rhodochrous ATCC 4275 (Nocardia corallina) has a restriction-modification system with the same recognition sequence, methylation site and cleavage site as the SalI restriction-modification system. Both the restriction endonuclease and the DNA-methyltransferase (DNA-MTase) have been partially purified and characterized. The nuclease has requirements of activity similar to SalI, and a native Mr of about 46,000. The DNA-MTase is a protein with an Mr of about 67,000. No DNA homology was detected between the cloned salI restriction-modification genes of Streptomyces albus and R. rhodochrous chromosomal DNA.

Effects of sinefungin and S-adenosylhomocysteine on DNA and protein methyltransferases from Streptomyces and other bacteria.

Sinefungin is a naturally occurring nucleoside isolated from cultures of Streptomyces griseolus and S. incarnatus. It is structurally related to S-adenosyl-methionine (SAM) and S-adenosyl-L-homocysteine (SAH). Its effect and level of action on prokaryotes has not been studied with the same detail as with eukaryotic cells. In this report we describe the effect of sinefungin and SAH on several Streptomyces methyltransferases (DNA and protein MTases) and on other bacterial DNA-MTases. Protein MTases are resistant to sinefungin, whereas DNA-MTases are inhibited. Adenine MTases however, seem more sensitive to this analogue than cytosine MTases.