tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.

tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.

A taxonomic study was made of the genus Leuconostoc. The species in the genus were divided into three subclusters by phylogenetic analysis based on the 16S rRNA gene sequences. The three subclusters were the Leuconostoc mesenteroides subcluster (comprising L. carnosum, L. citreum, L. gasicomitatum, L. gelidum, L. inhae, L. kimchii, L. lactis, L. mesenteroides and L. pseudomesenteroides), the L. fructosum subcluster (L. durionis, L. ficulneum, L. fructosum and L. pseudoficulneum) and the L. fallax subcluster (L. fallax). Phylogenetic trees based on the sequences of the 16S-23S rRNA gene intergenic spacer region, the rpoC gene or the recA gene indicated a good correlation with the phylogenetic tree based on 16S rRNA gene sequences. The species in the L. fructosum subcluster were morphologically distinguishable from the species in the L. mesenteroides subcluster and L. fallax as species in the L. fructosum subcluster had rod-shaped cells. In addition, the four species in the L. fructosum subcluster needed an electron acceptor for the dissimilation of d-glucose and produced acetic acid from d-glucose rather than ethanol. On the basis of evidence presented in this study, it is proposed that the four species in the L. fructosum subcluster, Leuconostoc durionis, Leuconostoc ficulneum, Leuconostoc fructosum and Leuconostoc pseudoficulneum, should be transferred to a novel genus, Fructobacillus gen. nov., as Fructobacillus durionis comb. nov. (type strain D-24(T)=LMG 22556(T)=CCUG 49949(T)), Fructobacillus ficulneus comb. nov. (type strain FS-1(T)=DSM 13613(T)=JCM 12225(T)), Fructobacillus fructosus comb. nov. (type strain IFO 3516(T)=DSM 20349(T)=JCM 1119(T)=NRIC 1058(T)) and Fructobacillus pseudoficulneus comb. nov. (type strain LC-51(T)=DSM 15468(T)=CECT 5759(T)). The type species of the genus Fructobacillus is Fructobacillus fructosus gen. nov., comb. nov.. No significant physiological and biochemical differences were found between the species in the L. mesenteroides subcluster and L. fallax in the present study and thus L. fallax remains as a member of the genus Leuconostoc.

Undefined mesophilic mixed (DL-type) starter cultures are composed of predominantly Lactococcus lactis subspecies and 1-10% Leuconostoc spp. The composition of the Leuconostoc population in the starter culture ultimately affects the characteristics and the quality of the final product. The scientific basis for the taxonomy of dairy relevant leuconostocs can be traced back 50 years, and no documentation on the genomic diversity of leuconostocs in starter cultures exists. We present data on the Leuconostoc population in five DL-type starter cultures commonly used by the dairy industry. The analyses were performed using traditional cultivation methods, and further augmented by next-generation DNA sequencing methods. Bacterial counts for starter cultures cultivated on two different media, MRS and MPCA, revealed large differences in the relative abundance of leuconostocs. Most of the leuconostocs in two of the starter cultures were unable to grow on MRS, emphasizing the limitations of culture-based methods and the importance of careful media selection or use of culture independent methods. Pan-genomic analysis of 59 Leuconostoc genomes enabled differentiation into twelve robust lineages. The genomic analyses show that the dairy-associated leuconostocs are highly adapted to their environment, characterized by the acquisition of genotype traits, such as the ability to metabolize citrate. In particular, Leuconostoc mesenteroides subsp. cremoris display telltale signs of a degenerative evolution, likely resulting from a long period of growth in milk in association with lactococci. Great differences in the metabolic potential between Leuconostoc species and subspecies were revealed. Using targeted amplicon sequencing, the composition of the Leuconostoc population in the five commercial starter cultures was shown to be significantly different. Three of the cultures were dominated by Ln. mesenteroides subspecies cremoris. Leuconostoc pseudomesenteroides dominated in two of the cultures while Leuconostoc lactis, reported to be a major constituent in fermented dairy products, was only present in low amounts in one of the cultures. This is the first in-depth study of Leuconostoc genomics and diversity in dairy starter cultures. The results and the techniques presented may be of great value for the dairy industry.

Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.

A mouse testis cDNA expression library (Clontech) was screened with a synthetic oligonucleotide ligand containing CT-rich motifs derived from the rat skeletal muscle actin gene promoter. These motifs bind nuclear proteins, and seem to be involved in the regulation of the gene. Analysis of isolated clones, which expressed proteins that specifically bind the oligonucleotide, indicated that they were derived from a single gene. This gene was identified as a contaminant of bacterial origin (Leuconostoc lactis). The cloned gene from L. lactis encodes a protein with significant homology to bacterial ribosomal protein S1, which we designated LrpS1-L. Band shift analysis and competition experiments indicated that both the bacterial protein and a mouse nuclear protein specifically bind to the same CT-rich motif of the skeletal muscle actin promoter. Furthermore, antibodies against the recombinant bacterial protein interfered with the formation of complex between the CT-rich element and the mouse nuclear protein. These results indicate that the bacterial LrpS1-L protein and the mammalian protein bind the same CT-rich motif and share common antigenic epitopes.

One cDNA clone (G7) was isolated from a lambda gt11 human liver cDNA library by the reaction with a serum containing anti-dsDNA Abs and was ligated into pGEX-1 lambda T vector. All the 10 SLE sera with anti-dsDNA, 2 samples of human monoclonal anti-dsDNA (33.C9 and 33.H11), and 2 affinity-purified anti-dsDNA Abs recognized the glutathione S-transferase fusion protein expressed by G7 (G7-FP). Ab binding to the recombinant protein expressed by G7 (G7-RP) and to G7-FP was inhibited completely by calf thymus dsDNA. The cDNA was 1314 nucleotides in length and contained an open reading frame encoding 352 amino acids. However, it seems to be a partial length cDNA because the affinity-purified Ab from G7-FP recognized only a 104-kDa protein on Western blot using MOLT4 cell extract. The nucleotide sequence of G7 was homologous (99% identity) to a cDNA encoding human ribosomal protein (r-protein) S1 homologue mRNA. The encoded protein contains repeating residues as a feature of r-proteins S1. Cytoplasmic and nucleolar staining of 33.H11 on indirect immunofluorescence (IF) using HEP 2 cells was inhibited by both G7-RP and calf thymus dsDNA. On ELISA, 33.H11 had a higher affinity for G7-RP than for DNA while 33.C9 had a higher affinity for DNA than for G7-RP and binds nuclei on IF. We conclude that G7 encodes a portion of human r-protein S1 and anti-dsDNA Abs cross-react with this protein.

The nucleotide sequence of the Leuconostoc lactis 533 cryptic plasmid pCI411 (2926 bp) was determined. Analysis revealed the presence of three open reading frames (ORFs). ORF 1 was capable of encoding a 24.9 kDa peptide which shared homology with the replication initiation protein (RepB) from a number of Gram-positive rolling circle plasmids. ORF 2 could encode a peptide of 6.6 kDa which was homologous to the RepC protein of the lactococcal plasmid pWV01. A function could not be assigned to ORF 3, which was capable of encoding a 12.1 kDa peptide. Transcription-translation analysis indicated the presence of three peptides of the predicted molecular masses. A putative double strand origin of replication (DSO) was identified which showed strong similarity with the DSO of a number of Gram-positive plasmids including pE194 from Staphylococcus. Structural analysis identified a number of direct and indirect repeats in addition to putative recombination-specific sites (RSA and RSB) in the non-coding region of pCI411. The observed characteristics suggest that this plasmid replicates using the rolling circle mechanism. pCI411, which could be introduced into Leuconostoc, Lactococcus, Streptococcus, Lactobacillus and Bacillus is the first plasmid from the genus Leuconostoc to be characterized in such detail.

The gene encoding the lactose transport protein (lacS) of Leuconostoc lactis NZ6009 has been cloned from its native lactose plasmid, pNZ63, by functional complementation of lactose permease-deficient Escherichia coli mutants. Nucleotide sequence analysis revealed an open reading frame with the capacity to encode a protein of 639 amino acids which had limited but significant identity to the lactose transport carriers (LacS) of Streptococcus thermophilus (34.5%) and Lactobacillus bulgaricus (35.6%). This similarity was present both in the amino-terminal hydrophobic carrier domain, which is homologous to the E. coli melibiose transporter, and in the carboxy-terminal enzyme IIA-like regulatory domain. The flanking regions of DNA surrounding lacS were also sequenced. Preceding the lacS gene was a small open reading frame in the same orientation encoding a deduced 95-amino-acid protein with a sequence similar to the amino-terminal portion of beta-galactosidase I from Bacillus stearothermophilus. The lacS gene was separated from the downstream beta-galactosidase genes (lacLM) by 2 kb of DNA containing an IS3-like insertion sequence, which is a novel arrangement for lac genes in comparison with that in other lactic acid bacteria. The lacS gene was cloned in an E. coli-Streptococcus shuttle vector and was expressed both in a lacS deletion derivative of S. thermophilus and in a pNZ63-cured strain, L. lactis NZ6091. The role of the LacS protein was confirmed by uptake assays in which substantial uptake of radiolabeled lactose or galactose was observed with L. lactis or S. thermophilus plasmids harboring an intact lacS gene. Furthermore, galactose uptake was observed in NZ6091, suggesting the presence of at least one more transport system for galactose in L. lactis.

We report the cloning of a human complementary DNA that encodes a protein which exhibits 36% identity and 62% similarity to Escherichia coli ribosomal protein S1 (rpS1), including conservation of four copies of an RNA-binding domain. This clone was obtained by ligand-screening a lambda gt11 expression library with a DNA probe derived from the CYBB gene promoter. Electrophoretic mobility shift and Southwestern blot assays confirm DNA binding activity of the protein, which exhibits preferential binding to single-stranded and double-stranded DNA and a low binding affinity for RNA. Hence, the rpS1 protein domain previously identified as an RNA-binding motif can also serve as a DNA-binding domain.

A novel insertion sequence, designated IS1070, was identified on the lactose plasmid of Leuconostoc lactis NZ6009 by nucleotide sequence analysis. The 1027-bp sequence contains partially matched (24 of 28 bp) inverted repeats and has one long open reading frame. The deduced 305-amino-acid sequence demonstrated homology to transposases of IS30 from Escherichia coli, IS4351 from Bacteroides fragilis, IS1086 from Alcaligenes eutrophus, IS1161 from Streptococcus salivarius, ISAS2 from Aeromonas salmonicida and a putative protein encoded by ORF3 of virus SpV1-R8A2 B from Spiroplasma citri. At least fifteen IS1070-like sequences were detected in the genome of the parent Lc. lactis strain and five of these were situated on plasmids. Analysis of the direct repeats of two of these copies with that of IS1070 revealed differences in the target duplication lengths.

The citrate permease determinant (citP) in several Leuconostoc strains was demonstrated to be plasmid encoded by curing experiments and hybridization studies with a DNA fragment containing the citP gene from Lactococcus lactis subsp. lactis biovar diacetylactis NCDO176. Cloning and nucleotide sequence analysis of Leuconostoc lactis NZ6070 citP revealed almost complete identity to lactococcal citP.