Genetic Evidence for a Defective Xylan Degradation Pathway in Lactococcus lactis

Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains, Lactococcus lactis 210, L. lactis IO-1, and L. lactis NRRL B-4449. Immediately downstream of the xylulose kinase gene (xylB) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl. Environ. Microbiol. 66:3974–3980, 1999) are two open reading frames encoding a mutarotase (xylM) and a xyloside transporter (xynT) and a partial open reading frame encoding a β-xylosidase (xynB). These are functions previously unreported for lactococci or lactobacilli. The mutarotase activity of the putative xylM gene product was confirmed by overexpression of the L. lactis enzyme in Escherichia coli and purification of recombinant XylM. We hypothesize that the mutarotase links xylan degradation to xylose metabolism due to the anomeric preference of xylose isomerase. In addition, Northern hybridization experiments suggested that the xylM and xynTB genes are cotranscribed with the xylRAB genes, responsible for xylose metabolism. Although none of the three strains appeared to metabolize xylan or xylobiose, they exhibited xylosidase activity, and L. lactis IO-1 and L. lactis NRRL B-4449 had functional mutarotases.     

Dissolution of xylose metabolism in Lactococcus lactis

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl(-). Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl(+) strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose.     

Dissolution of Xylose Metabolism in Lactococcus lactis

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl⁻. Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl⁺ strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose.     

Complete genome sequence of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid

We report the complete genome sequence of Lactococcus lactis IO-1 (= JCM7638). It is a nondairy lactic acid bacterium, produces nisin Z, ferments xylose, and produces predominantly L-lactic acid at high xylose concentrations. From ortholog analysis with other five L. lactis strains, IO-1 was identified as L. lactis subsp. lactis.     

Restoration of a defective Lactococcus lactis xylose isomerase

The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp. lactis strains, 210 (Xyl(-)) and IO-1 (Xyl(+)), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated. The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs. The purified IO-1 XI was soluble with K(m) and k(cat) being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive. Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble. The K(m) and k(cat) of this mutant were 4.15 mM and 141/s, respectively. One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI. The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility. The dissolution of XI activity in L. lactis subsp. lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.     

Gene encoding a dextransucrase-like protein in Leuconostoc mesenteroides NRRL B-512F

A gene, dsrT, encoding a dextransucrase-like protein was isolated from the genomic DNA libraries of Leuconostoc mesenteroides NRRL B-512F dextransucrase-like gene. The gene was similar to the intact open reading frames of the dextransucrase gene dsrS of L. mesenteroides NRRL B-512F, dextransucrase genes of strain NRRL B-1299 and streptococcal glucosyltransferase genes, but was truncated after the catalytic domain, apparently by the deletion of five nucleotides. dsrT mRNA was produced in this strain L. mesenteroides when cells were grown in a sucrose medum, but at a level of 20% of that of dsrS mRNA. The molecular weight of the dsrT gene product was 150,000 by SDS-PAGE. The product did not synthesize dextran, but had weak sucrose cleaving activity. The insertion of five nucleotides at the putative deletion point in dsrT resulted in an enzyme with a molecular weight of 210,000 and with dextransucrase activity.     

Cloning, characterization, and functional expression of the Klebsiella oxytoca xylodextrin utilization operon (xynTB) in Escherichia coli

Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose. Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E. coli. The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43. The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na(+)/melibiose symporters and related proteins. These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K. oxytoca and recombinant E. coli. Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E. coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons. Based on similarities in protein sequence, the xynTB genes in K. oxytoca appear to have originated from a gram-positive ancestor related to L. lactis. Functional expression of xynB allowed ethanologenic E. coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements. 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase. The rate of xylodextrin utilization by recombinant E. coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport. Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate. Xylodextrins were utilized more rapidly by recombinant E. coli than K. oxytoca M5A1 (the source of xynT and xynB). XynB exhibited weak arabinosidase activity, 3% that of xylosidase.     

Glucose repression of xylose utilisation by Lactococcus lactisIO-1

In dual substrate (5 g glucose l , 5 g xylose l ) batch fermentation of L. lactis IO-1 a classic diauxie was observed. In batch fermentations (5 g xylose l ) xylose isomerase activity was only detected in xylose grown cells. In mixed-substrate, carbon limited chemostat cultures (5 g glucose l , 5 g xylose l ) xylose utilisation was partially repressed by glucose at dilution rates above 0.01 h and completely repressed at 0.50 h .     

Deciphering the function of an ORF: Salmonella enterica DeoM protein is a new mutarotase specific for deoxyribose

We identified in Salmonella enterica serovar Typhi a cluster of four genes encoding a deoxyribokinase (DeoK), a putative permease (DeoP), a repressor (DeoQ), and an open reading frame encoding a 337 amino acid residues protein of unknown function. We show that the latter protein, called DeoM, is a hexamer whose synthesis is increased by a factor over 5 after induction with deoxyribose. The CD spectrum of the purified recombinant protein indicated a dominant contribution of betatype secondary structure and a small content of alpha-helix. Temperature and guanidinium hydrochloride induced denaturation of DeoM indicated that the hexamer dissociation and monomer unfolding are coupled processes. DeoM exhibits 12.5% and 15% sequence identity with galactose mutarotase from Lactococcus lactis and respectively Escherichia coli, which suggested that these three proteins share similar functions. Polarimetric experiments demonstrated that DeoM is a mutarotase with high specificity for deoxyribose. Site-directed mutagenesis of His183 in DeoM, corresponding to a catalytically active residue in GalM, yielded an almost inactive deoxyribose mutarotase. DeoM was crystallized and diffraction data collected for two crystal systems, confirmed its hexameric state. The possible role of the protein and of the entire gene cluster is discussed in connection with the energy metabolism of S. enterica under particular growth conditions.     

Xylanolytic Activity of Clostridium acetobutylicum

Of 20 strains of Clostridium spp. screened, 17 hydrolyzed larch wood xylan. Two strains of Clostridium acetobutylicum, NRRL B527 and ATCC 824, hydrolyzed xylan but failed to grow on solid media with larch xylan as the sole carbon source; however, strain ATCC 824 was subsequently found to grow on xylan under specified conditions in a chemostat. These two strains possessed cellulolytic activity and were therefore selected for further studies. In cellobiose-limited continuous cultures, strain NRRL B527 produced maximum xylanase activity at pH 5.2. Strain ATCC 824 produced higher xylanase, xylopyranosidase, and arabinofuranosidase activities in chemostat culture with xylose than with any other soluble carbon source as the limiting nutrient. The activities of these enzymes were markedly reduced when the cells were grown in the presence of excess glucose. The xylanase showed maximum activity at pH 5.8 to 6.0 and 65°C. The enzyme was stable on the alkaline side of pH 5.2 but was unstable below this pH value. The extracellular xylanolytic activity from strain ATCC 824 hydrolyzed 12% of the larch wood xylan during a 24-h incubation period, yielding xylose, xylobiose, and xylotriose as the major hydrolysis products. Strain ATCC 824, after being induced to grow in batch culture in xylan medium supplemented with a low concentration of xylose, failed to grow reproducibly in unsupplemented xylan medium. A mutant obtained by mutagenesis with ethyl methanesulfonate was able to grow reproducibly in batch culture on xylan. Both the parent strain and the mutant were able to grow with xylan as the sole source of carbohydrate in continuous culture with the pH maintained at either 5.2 or 6.0. Under these conditions, the cells utilized approximately 50% of the xylan.     

Heteropolysaccharide Formation by Arthrobacter viscosus Grown on Xylose and Xylose Oligosaccharides

Arthrobacter viscosus NRRL B-1973 produces its viscous extracellular polysaccharides (EPS) when grown on media containing xylose or enzymatic xylan hydrolysates. Crude EPS formation from xylose averaged 12 g/liter when initial culture pH was adjusted to 8.0 and total nitrogen was limited to 0.03%. Purified EPS from pentose and hexose substrates were analyzed for their monosaccharide, acetyl, and uronic acid components, intrinsic viscosities, and average molecular masses. Differences were apparent in degrees of acetylation, molecular masses, and intrinsic viscosities of the heteropolysaccharides produced on different carbon sources.     

Two different pathways for D-xylose metabolism and the effect of xylose concentration on the yield coefficient of L-lactate in mixed-acid fermentation by the lactic acid bacterium Lactococcus lactis IO-1

In lactic acid bacteria, pentoses are metabolized via the phosphoketolase pathway, which catalyzes the cleavage of D-xylulose-5-phosphate to equimolar amounts of glyceraldehyde 3-phosphate and acetylphosphate. Hence the yield coefficient of lactate from pentose does not exceed 1.0 mol/mol, while that of Lactococcus lactis IO-1(JCM7638) at high D-xylose concentrations often exceeds the theoretical value. This suggests that, in addition to the phosphoketolase pathway, L. lactisIO-1 may possess another metabolic pathway that produces only lactic acid from xylose. In the present study, the metabolism of xylose in L. lactisIO-1 was deduced from the product formation and enzyme activities of L. lactisIO-1 in batch culture and continuous culture. During cultivation with xylose concentrations above ca. 50 g/l, the yield coefficient of L-lactate exceeded 1.0 mol/mol while those of acetate, formate and ethanol were very low. At xylose concentrations less than 5 g/l, acetate, formate and ethanol were produced with yield coefficients of about 1.0 mol/mol, while L-lactate was scarcely produced. In cells grown at high xylose concentrations, a marked decrease in the specific activities of phosphoketolase and pyruvate formate lyase (PFL), and an increase in those of transketolase and transaldolase were observed. These results indicate that in L. lactisIO-1 xylose may be catabolized by two different pathways, the phosphoketolase pathway yielding acetate, formate and ethanol, and the pentose phosphate (PP)/glycolytic pathway which converts xylose to L-lactate only. Furthermore, it was deduced that the change in the xylose concentration in the culture medium shifts xylulose 5-phosphate metabolism between the phosphoketolase pathway and the PP/glycolytic pathway in L. lactisIO-1, and pyruvate metabolism between cleavage to acetyl-CoA and formic acid by PFL and the reduction to L-lactate by lactate dehydrogenase.     

Cloning and verification of the Lactococcus lactis pyrG gene and characterization of the gene product, CTP synthase

The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactis strains harboring disrupted pyrG alleles were constructed. These mutants required cytidine for growth, proving that in L. lactis, the pyrG product is the only enzyme responsible for the amination of UTP to CTP. In contrast to the situation in Escherichia coli, an L. lactis pyrG mutant could be constructed in the presence of a functional cdd gene encoding cytidine deaminase. A characterization of the enzyme revealed similar properties as found for CTP synthases from other organisms. However, unlike the majority of CTP synthases the lactococcal enzyme can convert dUTP to dCTP, although a half saturation concentration of 0.6 mm for dUTP makes it unlikely that this reaction plays a significant physiological role. As for other CTP synthases, the oligomeric structure of the lactococcal enzyme was found to be a tetramer, but unlike most of the other previously characterized enzymes, the tetramer was very stable even at dilute enzyme concentrations.     

Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene.

A new bacteriocin, termed lactococcin A (LCN-A), from Lactococcus lactis subsp. cremoris LMG 2130 was purified and sequenced. The polypeptide contained no unusual amino acids and showed no significant sequence similarity to other known proteins. Only lactococci were killed by the bacteriocin. Of more than 120 L. lactis strains tested, only 1 was found resistant to LCN-A. The most sensitive strain tested, L. lactis subsp. cremoris NCDO 1198, was inhibited by 7 pM LCN-A. By use of a synthetic DNA probe, lcnA was found to be located on a 55-kb plasmid. The lcnA gene was cloned and sequenced. The sequence data revealed that LCN-A is ribosomally synthesized as a 75-amino-acid precursor including a 21-amino-acid N-terminal extension. An open reading frame encoding a 98-amino-acid polypeptide was found downstream of and in the same operon as lcnA. We propose that this open reading frame encodes an immunity function for LCN-A. In Escherichia coli lcnA did not cause an LCN-A+ phenotype. L. lactis subsp. lactis IL 1403 produced small amounts of the bacteriocin and became resistant to LCN-A after transformation with a recombinant plasmid carrying lcnA. The other lactococcal strains transformed with the same recombinant plasmid became resistant to LCN-A but did not produce any detectable amount of the bacteriocin.     

Antisense mRNA-Mediated Bacteriophage Resistance in Lactococcus lactis subsp. lactis

Resistance to a broad class of isometric bacteriophages that infect strains of Lactococcus lactis has been engineered into a dairy starter by expression of antisense mRNA targeted against a conserved bacteriophage gene. Maximum protection is obtained only when the entire 1,654-bp coding sequence for a 51-kDa protein is positioned in the antisense orientation with respect to a promoter sequence that functions in L. lactis subsp. lactis. Expression of the antisense mRNA results in more than 99% reduction of the total number of PFU. Plaques that do form are characterized by their relatively small size and irregular shape. A variety of truncated genes, including the open reading frame expressed in the sense orientation, fail to provide any significant measure of resistance as compared with that of the intact open reading frame. Southern hybridization with probes specific for the conserved region reveal that the [ill] plasmid constructs are maintained despite the presence of a large complement of other indigenous plasmids. Strains harboring the antisense mRNA plasmid construct grow and produce acid at a rate equivalent to that of the host strain alone, suggesting that antisense expression is not deleterious to normal cellular metabolism.     

The pyrH gene of Lactococcus lactis subsp. cremoris encoding UMP kinase is transcribed as part of an operon including the frr1 gene encoding ribosomal recycling factor 1

The pyrH gene of Lactococcus lactis subsp. cremoris MG1363, encoding UMP kinase, has been sequenced and cloned. It encodes a polypeptide of 239 amino acid residues (deduced molecular weight of 25951), which was shown to complement a temperature sensitive pyrH mutation in Escherichia coli, thus establishing the ability of the encoded protein to synthesize UDP. The pyrH gene in L. lactis is flanked downstream by frr1 encoding ribosomal recycling factor 1 and upstream by an open reading frame, orfA, of unknown function. The three genes were shown to constitute an operon transcribed in the direction orfA-pyrH-frr1 from a promoter immediately in front of orfA. This operon belongs to an evolutionary highly conserved gene cluster, since the organization of pyrH on the chromosomal level in L. lactis shows a high resemblance to that found in Bacillus subtilis as well as in Escherichia coli and several other prokaryotes     

The Hypocrea jecorina gal10 (uridine 5'-diphosphate-glucose 4-epimerase-encoding) gene differs from yeast homologues in structure,genomic organization and expression

As part of a comprehensive study on lactose metabolism in Hypocrea jecorina (anamorph: Trichoderma reesei), a genomic clone of the gal10 gene encoding H. jecorina uridine 5'-diphosphate (UDP)-glucose 4-epimerase has been cloned and sequenced. It contains an open reading frame of 1548-base pair, interrupted by three introns, and encoding a 370-amino acids protein with similarity to pro- and eukaryotic UDP-glucose-4-epimerases. H. jecorina Gal10 does not contain the C-terminal mutarotase domain which is present in yeast Gal10 proteins but is able to functionally complement a corresponding Saccharomyces cerevisiae gal10 mutant. gal10 is not clustered with other H. jecorina gal genes (gal7, gene encoding galactose-1-phosphate uridylyltransferase and gal1, gene encoding galactokinase). The genomic location of H. jecorina gal10 and gal7 was syntenic with that in Neurospora crassa and colinear over an area of 6 and 3.5-kilobase. gal10 is constitutively expressed, and--unlike H. jecorina gal7--not further stimulated by D-galactose or L-arabinose or its corresponding polyols.     

Mannitol production by lactic acid bacteria grown in supplemented carob syrup

Detailed kinetic and physiological characterisation of eight mannitol-producing lactic acid bacteria, Leuconostoc citreum ATCC 49370, L. mesenteroides subsp. cremoris ATCC19254, L. mesenteroides subsp. dextranicum ATCC 19255, L. ficulneum NRRL B-23447, L. fructosum NRRL B-2041, L. lactis ATCC 19256, Lactobacillus intermedius NRRL 3692 and Lb. reuteri DSM 20016, was performed using a carob-based culture medium, to evaluate their different metabolic capabilities. Cultures were thoroughly followed for 30 h to evaluate consumption of sugars, as well as production of biomass and metabolites. All strains produced mannitol at high yields (>0.70 g mannitol/g fructose) and volumetric productivities (>1.31 g/l h), and consumed fructose and glucose simultaneously, but fructose assimilation rate was always higher. The results obtained enable the studied strains to be divided mainly into two groups: one for which glucose assimilation rates were below 0.78 g/l h (strains ATCC 49370, ATCC 19256 and ATCC 19254) and the other for which they ranged between 1.41 and 1.89 g/l h (strains NRRL B-3692, NRRL B-2041, NRRL B-23447 and DSM 20016). These groups also exhibited different mannitol production rates and yields, being higher for the strains with faster glucose assimilation. Besides mannitol, all strains also produced lactic acid and acetic acid. The best performance was obtained for L. fructosum NRRL B-2041, with maximum volumetric productivity of 2.36 g/l h and the highest yield, stoichiometric conversion of fructose to mannitol.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.