Extracellular material from Leuconostoc mesenteroides subsp. dextranicum mediates the aggregation of lactococcal cells: implications for the isolation of single strains

Cultures of Lactococcus lactis subsp. cremoris , originally derived from mixed cheese starter cultures, were assessed as pure following single colony selection and subculturing, yet nevertheless gave rise, under stress conditions, to an isolate with the ability to ferment citrate. The isolate was characterized with respect to its citrate enzymology and lactose fermentation and was identified as Leuconostoc mesenteroides subsp. dextranicum and assigned the strain number 663. The extracellular material (ECM) from Leuc. mesenteroides subsp. dextranicum 663 was characterized and found to contain carbohydrate, protein and phosphate (30.9, 50.7 and 18.4%, by weight, respectively). Glucose was the most prominent sugar (39% by weight of total carbohydrate) with mannose, galactose and rhamnose being the other major monosaccharides. The ECM protein resolved into a large number of bands on SDS-polyacrylamide gel electrophoresis, the most prominent having molecular masses of 40 and 49 kDa. The ECM from Leuc. mesenteroides subsp. dextranicum 663 caused aggregation of suspensions of lactococcal cells and may facilitate intergeneric interactions and/or co-culture during cheese starter strain isolation.     

Selection of Galactose-Fermenting Streptococcus thermophilus in Lactose-Limited Chemostat Cultures

Stock cultures of Streptococcus thermophilus are essentially galactose negative (Gal). Although both galactose 1-phosphate uridyl transferase and uridine-5-diphospho-glucose 4-epimerase are present, suggesting that the genes for the Leloir pathway exist, cells cannot induce high levels of galactokinase. Therefore, galactose is largely excreted when cultures are grown on lactose, and most strains cannot be readily adapted to grow on free galactose. Gal cultures were grown in a chemostat under lactose limitation in which high concentrations of residual galactose were present. Under this selection pressure, Gal organisms eventually took over the culture with all four strains examined. Gal cells had induced galactokinase, and three of the four strains grew on free galactose with doubling times of 40 to 50 min. When Gal organisms were grown on lactose in batch culture, the galactose moiety was only partially utilized while lactose was still present. As lactose was exhausted, and catabolite repression was lifted, the Leloir pathway enzymes (especially galactokinase) were induced and the residual galactose fermented. Neither phospho-beta-galactosidase activity nor the enzymes of the d-tagatose 6-phosphate pathway were detected in S. thermophilus. In contrast to Streptococcus cremoris and Streptococcus lactis, fermentation was homolactic with galactose in batch cultures and with lactose limitation in the chemostat. When mixed Gal-Gal cultures were repeatedly transferred in milk, the Gal cells became the dominant cell type. The Gal phenotype of stock cultures probably reflects their prolonged maintenance in milk.     

Ribosyl-diphthamide: Confirmation of structure by fast atom bombardment mass spectrometry

Diphtheria toxin inactivates protein synthesis elongation factor 2 by attaching ADP-ribose to an unusual post-translational amino acid derivative, diphthamide, in the factor. Previously, we prepared ribosyl-diphthamide from the ADP-ribosyl-factor and proposed on the basis of NMR spectral analysis that it is 1-α-d-ribofuranosyl-2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine [N. J. Oppenheimer, and J. W. Bodley, (1981) J. Biol. Chem.256, 8579–8581 and op. cit.]. Now, using fast atom bomardment mass spectrometry, the intact cation of ribosyl-diphthamide has been observed in the gas phase. The theoretical mass of the structure proposed for ribosyl-diphthamide uniquely agrees with the observed mass of the inact cation of the compound to within 2 ppm. Collisional activation decomposition mass spectral analysis provided additional structural confirmation. Thus, although the compound has not been synthesized, all available evidence appears uniquely consistent with the structure of ribosyl-diphthamide previously proposed.     

Fast atom bombardment combined with tandem mass spectrometry for the determination of nucleosides

The positive and negative ion fast atom bombardment (FAB) mass spectra and fast atom bombardment collisionally activated decomposition (CAD) spectra of a series of nucleosides and two dinucleotides are reported. The nucleosides studied are substituted forms of guanosine, adenosine, nebularine, tubercidin, uridine, and related pyrimidines. The FAB and CAD data both contain similar information. The CAD spectra are found to provide some structural information not found in the FAB mass spectra. Tandem mass spectrometry also allows emphasis to be put on weak fragments which are either not observed in the FAB mass spectrum or are lost in the matrix ion signals.     

Galactose fermentation by Streptococcus lactis and Streptococcus cremoris: pathways, products, and regulation

All of the lactic streptococci examined except Streptococcus lactis ML8 fermented galactose to lactate, formate, acetate, and ethanol. The levels of pyruvate-formate lyase and lactate dehydrogenase were elevated and reduced, respectively, in galactose-grown cells compared with glucose- or lactose-grown cells. Reduced intracellular levels of both the lactate dehydrogenase activator (fructose, 1,6-diphosphate) and pyruvate-formate lyase inhibitors (triose phosphates) appeared to be the main factors involved in the diversion of lactate to the other products. S. lactis ML8 produced only lactate from galactose, apparently due to the maintenance of high intracellular levels of fructose 1,6-diphosphate and triose phosphates. The growth rates of all 10 Streptococcus cremoris strains examined decreased markedly with galactose concentrations below about 30 mM. This effect appeared to be correlated with uptake predominantly by the low-affinity galactose phosphotransferase system and initial metabolism via the D-tagatose 6-phosphate pathway. In contrast, with four of the five S. lactis strains examined, galactose uptake and initial metabolism involved more extensive use of the high-affinity galactose permease and Leloir pathway. With these strains the relative flux of galactose through the alternate pathways would depend on the exogenous galactose concentration.     

Influence of the lactose plasmid on the metabolism of galactose by Streptococcus lactis.

Streptococcus lactis strain DR1251 was capable of growth on lactose and galactose with generation times, at 30 degrees C, of 42 and 52 min, respectively. Phosphoenolpyruvate-dependent phosphotransferase activity for lactose and galactose was induced during growth on either substrate. This activity had an apparent K(m) of 5 x 10(-5) M for lactose and 2 x 10(-2) M for galactose. beta-d-Phosphogalactoside galactohydrolase activity was synthesized constitutively by these cells. Strain DR1251 lost the ability to grow on lactose at a high frequency when incubated at 37 degrees C with glucose as the growth substrate. Loss of ability to metabolize lactose was accompanied by the loss of a 32-megadalton plasmid, pDR(1), and Lac(-) isolates did not revert to a Lac(+) phenotype. Lac(-) strains were able to grow on galactose but with a longer generation time. Galactose-grown Lac(-) strains were deficient in beta-d-phosphogalactoside galactohydrolase activity and phosphoenolpyruvate phosphotransferase activity for both lactose and galactose. There was also a shift from a predominantly homolactic to a heterolactic fermentation and a fivefold increase in galactokinase activity, relative to the Lac(+) parent strain grown on galactose. These results suggest that S. lactis strain DR1251 metabolizes galactose primarily via the tagatose-6-phosphate pathway, using a lactose phosphoenolpyruvate phosphotransferase activity to transport this substrate into the cell. Lac(-) derivatives of strain DR1251, deficient in the lactose phosphoenolpyruvate phosphotransferase activity, appeared to utilize galactose via the Leloir pathway.     

DNA Recognition by Quinoline Antibiotics: Use of Base-Modified DNA Molecules to Investigate Determinants of Sequence-Specific Binding of Luzopeptin

Abstract

The luzopeptin antibiotics contain a cyclic decadepsipeptide to which are attached two quinoline chromophores that bisintercalate into DNA. Although they bind DNA less tightly than the structurally related quinoxaline antibiotics echinomycin and triostin A, the molecular basis of their interaction remains unclear. We have used the PCR in conjunction with novel nucleotides to create specifically modified DNA for footprinting experiments. In order to study the influence that removal, addition or relocation of the guanine 2-amino group, which normally identifies G. C base pairs from the minor groove, has on the interaction of luzopeptin antibiotics with DNA. The presence of a purine 2-amino group is not strictly required for binding of luzopeptin to DNA, but the exact location of this group can alter the position of preferred drug binding sites. It is, however, not the sole determinant of nucleotide sequence recognition in luzopeptin-DNA interaction. Nor can the selectivity of luzopeptin be attributed to the quinoline chromophores, suggesting that an analogue mode of DNA recognition may be operative. This is in contrast to the digital readout that seems to predominate with the quinoxaline antibiotics.