Aims: Five species of the Gram-positive bacterial genus Lactococcus (Lactococcus lactis, L. garvieae, L. plantarum, L. piscium and L. raffinolactis) are currently recognized. The aim of this work was to develop a simple approach for the identification of these species, as well as to differentiate the industrially important dairy subspecies L. lactis subsp. lactis and L. lactis subsp. cremoris. Methods and Results: Methods were devised based on specific polymerase chain reaction (PCR) amplifications that exploit differences in the sequences of the 16S ribosomal RNA genes of each species, followed by restriction enzyme cleavage of the PCR products. The techniques developed were used to characterize industrial cheese starter strains of L. lactis and the results were compared with biochemical phenotype and DNA sequence data. Conclusions: The PCR primers designed can be used simultaneously, providing a simple scheme for screening unknown isolates. Strains of L. lactis show heterogeneity in the 16S ribosomal RNA gene sequence. Significance and Impact of the Study: This work provides an integrated set of methods for differentiation and identification of lactococcal species associated with agricultural, veterinary, medical and processed food industries.

Arabinogalactan-proteins (AGPs) are proteoglycans containing a high proportion ofcarbohydrate (typically >90%) linked to a protein backbone rich in hydroxyproline (Hyp), Ala Approximately 93% of the Pro residues are hydroxylated and hence are potential sites for glycosylation.Arabinogalactan-proteins (AGPs) occur predominantly in the intercellular spaces of plant tissues but are also associated with membranes, some cytoplasmic organelles, and the cell wall (for reviews see refs. 1-5). AGPs bind to and are precipitated by the P-glucosyl Yariv reagent (6). The function of AGPs is not established, but they may be involved in development, cell-cell interactions, and plant defense.The carbohydrate component of AGPs is generally composed of arabinose and galactose with minor amounts of other sugars. Linkage analysis is consistent with a structure based on a 3-linked (3-galactosyl backbone, branched through C(0)6 to 6-linked galactosyl side chains. The arabinose is most often present as terminal residues. The protein is usually a minor component with characteristically high levels of hydroxyproline (Hyp), Ala, and Ser (for exceptions see refs. 7 and 8). Relatively little is known about the structure ofthe protein core ofAGPs; only a few peptide sequences are available (7,(9)(10)(11) AGPs were deglycosylated using anhydrous HF (18) and fractionated by size-exclusion FPLC and RP-HPLC according to Fig. 1 D-E. The protein backbones were digested with thermolysin, and the products were separated on a C18 microbore HPLC column (Ultrasphere ODS, 2.1 x 250 mm) and eluted with a gradient of acetonitrile in 0.1% aqueous TFA. Individual peaks were repurified and sequenced (19). Amino acid analyses were performed as described by Simpson et al. (20).Isolation of cDNA Clones. A 68-base oligonucleotide, 5'-GCAAAATCACCAACAGCAACACCACCAACAGCAA-CACCACCATCAGCAGTATATAGTGAGTCGTATTA-3', was synthesized. The first part of the sequence codes for the AGP peptide A-K-S-O-T-A-T-O-O-T-A-T-O-O-S-A-V (

SummaryThis paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan-proteins (AGPs), one from pear cell suspension cultures (AGPP¢2) and the other from suspension cultures of Nicotiana alata (AGPNa2). The proteins encoded by these cDNAs are quite different from the 'classical' AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N. alata styles. The cDNA for AGPP¢2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues. Asn residues are not a feature of the 'classical' AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids. The cDNA for AGPNa2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Sar, Ala, Thr and the other rich in Asn, Tyr and Sar. The composition and sequence of the Pro-rich domain resembles that of the 'classical' AGP backbone. The Asn-rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the "classical' AGPs. The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side-chains. It also shows that differential expression of genes encoding AGP protein backbones, as well

Nicotiana alata has a style-specific hydroxyproline-rich glycoprotein (the 120 kDa glycoprotein) which has properties of both extensins and AGPs [19, 20]. The 120 kDa glycoprotein is a soluble component in the extracellular matrix of the transmitting tract of styles where it accounts for ca. 9% of the total buffer-soluble protein. Here we describe the molecular cloning of a cDNA representing the gene NaPRP5 which encodes the backbone of the 120 kDa glycoprotein. Expression of mRNA is restricted to styles, consistent with observations on the distribution of the 120 kDa glycoprotein. Levels of accumulation of the transcript encoding the 120 kDa protein backbone are not altered significantly by pollination with either compatible or incompatible pollen. The protein backbone of the 120 kDa glycoprotein, as predicted by the cDNA sequence, is composed of three distinct domains. The sequence of these domains, together with linkage analysis of the carbohydrate component of the 120 kDa glycoprotein, allows predictions of the likely distribution of substituent glycosyl chains along the protein backbone. The similarity of the C-terminal domains of the 120 kDa glycoprotein and GaRSGP, the galactose-rich style glycoprotein of N. alata, is consistent with the two molecules sharing a common antigenic domain in their backbones [31]. The sharing of domains between distinct hydroxyproline-rich glycoproteins suggests that identification of a glycoprotein of this class solely by its protein or carbohydrate epitope is not valid.

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