Lactose and galactose uptake by genetically engineered Pediococcus species

The ability to utilize lactose is requisite for lactic acid bacteria used as starters in the dairy industry. Modern genetic recombination techniques have facilitated the introduction of the lactose-positive phenotype into bacteria such as Pediococcus species, which traditionally have not been used as dairy starters. This study investigated lactose and galactose uptake along with phospho-β-galactosidase activity in pediococci that had been transformed with a Latococcus lactis lactose plasmid. Lactose-positive transformants, Pediococcus acidilactici SAL and Pediococcus pentosaceus SPL-2, demonstrated an ability to accumulate [¹⁴C]lactose at a rate greater than the Lactococcus lactis control. Phospho-β-galactosidase activity was also higher in transformants versus Lactococcus lactis. Studies of [³H]galactose uptake suggested that a wild-type galactose transport system and the introduced lactose phosphotransferase system both functioned in galactose uptake by Pediococcus spp. transformants. Significantly lower levels of free galactose were detected in milk fermented with Lactobacillus helveticus LH100 and SAL or SPL-2 than in milk fermented with a LH100 plus Streptococcus thermophilus TA061 control starter blend. Received: 16 September 1997 /  Received revision: 11 November 1997 / Accepted: 21 November 1997     

Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides

Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5α derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, β-casein (β-CN) (f193-209) and α_S1-casein (α_S1-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards α_S1-CN (f1-9) and β-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze β-CN (f193-209) and α_S1-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides α_S1-CN (f1-9), α_S1-CN (f1-13), and α_S1-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with β-CN (f193-209) and α_S1-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.     

Conversion of Lactococcus lactis cell envelope proteinase specificity by partial allele exchange

AIMS: To determine whether conversion of lactocepin substrate binding regions by gene replacement can alter lactocepin specificity in Lactococcus lactis starter bacteria without affecting other important strain properties. METHODS AND RESULTS: We utilized two-step gene replacement to convert substrate-binding determinants in the L. lactis prtP genes encoding group h (bitter) lactocepin in two industrial strains into the corresponding group b (nonbitter) variant. Analysis of lactocepin activity toward alpha(s1)-casein (f 1-23) by reversed-phase high-pressure liquid chromatography demonstrated enzyme specificity among isogenic derivatives had been altered in a manner that was consistent with predicted amino acid substitutions in substrate binding regions. Milk acidification properties of some mutants were not statistically different (P > 0.05) from wild-type parent strains, and strain propensity for autolysis was also not significantly (P > 0.05) changed. CONCLUSIONS: Conversion of lactocepin substrate binding regions by allele exchange can effectively alter lactocepin specificity in industrial strains of L. lactis without significantly affecting other important strain properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Methodology outlined in this study can be used to alter lactocepin specificity in commercial starter cultures with a propensity for bitter flavour defect, and prtP derivatives developed by this approach should be suitable for commercial application.     

An anterior signalling centre in Xenopus revealed by the homeobox gene XHex.

BACKGROUND: Signals from anterior endodermal cells that express the homeobox gene Hex initiate development of the most rostral tissues of the mouse embryo. The dorsal/anterior endoderm of the Xenopus gastrula, which expresses Hex and the putative head-inducing gene cerberus, is proposed to be equivalent to the mouse anterior endoderm. Here, we report the origin and signalling properties of this population of cells in the early Xenopus embryo. RESULTS: Xenopus anterior endoderm was found to derive in part from cells at the centre of the blastocoel floor that express XHex, the Xenopus cognate of Hex. Like their counterparts in the mouse embryo, these Hex-expressing blastomeres moved to the dorsal side of the Xenopus embryo as gastrulation commenced, and populated deep endodermal adjacent to Spemann's organiser. Experiments involving the induction of secondary axes confirmed that XHex expression was associated with anterior development. Ventral misexpression of XHex induced ectopic cerberus expression and conferred anterior signalling properties to the endoderm. Unlike the effect of misexpressing cerberus, these signals could not neuralise overlying ectoderm. CONCLUSIONS: XHex expression reveals the unexpected origin of an anterior signalling centre in Xenopus, which arises in part from the centre of the blastula and localises to the deep endoderm adjacent to Spemann's organiser. Signals originating from these endodermal cells impart an anterior identity to the overlying ectoderm, but are insufficient for neural induction. The anterior movement of Hex-expressing cells in both Xenopus and mouse embryos suggests that this process is a conserved feature of vertebrate development.     

Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida

Cattle, pig and sheep oocytes isolated from healthy cumulus-oocyte complexes were pooled, within species, to provide samples of immature denuded oocytes with intact zona pellucida (n = 1000 per sample) for determination of fatty acid mass and composition in total lipid, constituent phospholipid and triglyceride. Acyl-containing lipid extracts, transmethylated in the presence of a reference penta-decaenoic acid (15:0), yielded fatty acid methyl esters which were analysed by gas chromatograph. Mean (+/- SEM) fatty acid content in samples of pig oocytes (161 +/- 18 micrograms per 1000 oocytes) was greater than that in cattle (63 +/- 6 micrograms; P < 0.01) and sheep oocytes (89 +/- 7 micrograms; P < 0.05). Of 24 fatty acids detected, palmitic (16:0; 25-35%, w/w), stearic (18:0; 14-16%) and oleic (18:1n-9; 22-26%) acids were most prominent in all three species. Saturated fatty acids (mean = 45-55%, w/w) were more abundant than mono- (27-34%) or polyunsaturates (11-21%). Fatty acids of the n-6 series, notably linoleic (18:2n-6; 5-8%, w/w) and arachidonic acid (20:4n-6; 1-3%), were the most abundant polyunsaturates. Phospholipid consistently accounted for a quarter of all fatty acids in the three species, but ruminant oocytes had a lower complement of polyunsaturates (14-19%, w/w) in this fraction than pig oocytes (34%, w/w) which, for example, had a three- to fourfold greater linoleic acid content. An estimated 74 ng of fatty acid was sequestered in the triglyceride fraction of individual pig oocytes compared with 23-25 ng in ruminant oocytes (P < 0.01). It is concluded that the greater fatty acid content of pig oocytes is primarily due to more abundant triglyceride reserves. Furthermore, this species-specific difference, and that in respect of polyunsaturated fatty acid reserves, may underlie the contrasting chilling, culture and cryopreservation sensitivities of embryos derived from pig and ruminant (cattle, sheep) oocytes.     

Effective decisions and their verbal justification

Recent experimental work on human control of complex systems has drawn attention to the discrepancy that may exist between the person's reported knowledge of the system and their ability to control it. Sometimes people act correctly but cannot answer questions about what they are doing; sometimes they can say verbally what they should do (perhaps having had verbal instruction in the right answers), but still do not do it. This discrepancy is of major practical importance, for example in designing training programmes or in eliciting expert knowledge for incorporation in a mechanical 'expert system'. It is also puzzling for psychological theory, as it rules out certain plausible models of the functioning of the brain. This paper considers what mechanisms are still possible.