An integrative cloning vector was constructed using a randomly cloned HindIII-digested chromosomal fragment from Lactobacillus acidophilus ADH inserted into an Escherichia coli vector, pBluescript II SK+. Southern hybridization studies demonstrated homology of the inserted fragment with one other L. acidophilus strain and one Bifidobacterium strain. Identification of a SauI site located near the middle of the 1.9-kb ADH chromosomal fragment made it possible to clone the Lactobacillus bulgaricus beta-galactosidase (EC 3.2.1.23) gene into this vector. The vector was unable to replicate in the homologous host, L. acidophilus ADH, following electroporation. The chromosomal fragment allowed the integration of the beta-galactosidase gene (beta gal) into the host chromosome via homologous recombination. The size of the two flanking L. acidophilus ADH chromosomal fragments, approximately 0.95 kb each, was sufficient to allow the double cross-over to take place. Southern hybridization demonstrated that only L. acidophilus and L. bulgaricus DNA had been integrated into the chromosome of the host strain. The beta-galactosidase activity of the transformant was increased approximately 200-fold when compared to the enzyme activity of the wild-type strain. The beta gal gene remained stable in the transformant strain after 30 transfers in growth media without selection pressure. This first-generation integrative cloning vector is constructed solely of DNA from organisms consumed by humans and could be considered a food-grade vector system.

Seven laboratories participated in a WHO-sponsored international collaborative study, to evaluate methods for subtyping Listeria monocytogenes, by performing restriction fragment length polymorphism (RFLP) analysis-based subtyping of an international study set of 80 strains of L. monocytogenes that included 22 epidemiologically related groups. The RFLP analysis was done by Southern hybridization with one of two types of probes found in multiple copies on the chromosome of L. monocytogenes. Six laboratories performed ribotyping. These laboratories used EcoRI enzyme to restrict the L. monocytogenes DNA and ribosomal RNA or DNA as the probe for Southern hybridizations. The seventh laboratory used NciI to restrict the DNA. and two probes, one randomly cloned and the other containing repeat sequences cloned from L. monocytogenes DNA. The overall discriminating power of ribotyping, as estimated by calculation of Simpson's index of diversity, ranged from 0.83 to 0.88 for the six laboratories. The discriminating power of the combination of two probes used by Laboratory 7 was 0.91. Ribotyping and the cloned probes used by Laboratory 7 discriminated poorly between serotype 4b strains. Neither method identified three atypical strains (identified by other subtyping methods) included in three apparently epidemiologically related groups. Ribotyping did not discriminate between strains of serotypes 4b and 4b(X) in one epidemiologically related group of strains; one cloned probe used by Laboratory 7 discriminated between these strains. Intra-laboratory reproducibilities for the seven laboratories ranged from 80.0 to 100%, as determined by their abilities to correctly identify 11 pairs of duplicate strains included in the study set. Inter-laboratory reproducibilities were generally very good considering that no attempt was made to standardize protocols used by the participants.

Biotechnology promises to change the foods we eat. It will have an impact on the entire food chain, from the genetic improvement of key agricultural crops and farm animals to the processing, packaging and distribution of processed foods. What kinds of changes should we expect? How will these changes affect the food supply? How will future foods be regulated? Will the public accept biotechnology-derived foods?

The effect of nonfermented dairy products containing yogurt or acidophilus cultures on lactose utilization by lactose-maldigesting humans was investigated. Yogurt and acidophilus milk containing 10(7) or 10(8) of Streptococcus thermophilus and Lactobacillus bulgaricus, or Lactobacillus acidophilus, respectively, were prepared using commercially processed 2% low fat milk. Immediately following inoculation, products were refrigerated. Lactose maldigestion was monitored by measuring breath hydrogen excretion at hourly intervals for 8 h following consumption of 400 ml of each test meal containing approximately 20 g of lactose. The yogurt milk containing 10(8) cfu/ml was shown to contain significant concentrations of microbial beta-galactosidase (EC 3.2.1.23; approximately 3 U/ml), which remained stable for at least 14 d at refrigerator temperatures. Breath hydrogen peaks were delayed and significantly lower (approximately 20 ppm at 5 to 7 h) than control values (approximately 70 ppm at 4 h), and intolerance symptoms were eliminated in all subjects. Yogurt milk containing 10(7) cfu/ml demonstrated intermediate breath hydrogen values and was marginally significantly different from control values. Lactobacillus acidophilus strains with varying resistance to bile and total beta-galactosidase-producing potential were also tested. Only one strain, LA-1, which demonstrated low bile resistance and intermediate beta-galactosidase activity, was capable of significantly decreasing breath hydrogen values when 10(8) cfu/ml of milk was consumed.

The phage insensitivity mechanism of Streptococcus lactis ssp. diacetylactis KR2 was investigated. This strain harbored seven detectable plasmids ranging in size from 2.9 to 32 Mdaltons. Transformation of KR2 plasmid DNA into plasmid-free Streptococcus lactis LM0230 yielded lactose-positive transformants that were either sensitive or exhibited a reduced sensitivity to c2 phage. The latter possessed plasmids of 23.6 and 29 Mdaltons, designated pKR223 and pKR229, respectively. Lactose-positive phage-sensitive transformants contained only pKR229. Transformants exhibiting reduced phage sensitivity were cured of lactose-fermenting ability. Lactose-negative derivatives retained reduced phage sensitivity and possessed only pKR223, thus linking this phenotype to the 23.6Mdalton plasmid of KR2. The gene or genes responsible for the reduced phage sensitivity phenotype were cloned on a 17-kb HpalI fragment of pKR223 into the single HpalI sire on the streptococcal cloning vector, pGB301. The phage defense mechanism(s) encoded by pKR223 and the 17-kb fragment of pKR223 involved a reduction in plaque size, which appeared to be due to reduced phage burst size and abortive phage infection. The data provided evidence that the phage insensitivity phenotype