Control of replication of the Lactobacillus pentosus plasmid p353-2: evidence for a mechanism involving transcriptional attenuation of the gene coding for the replication protein.

The synthesis of plasmid DNA and of RNA encoded by the replication protein gene (rep) of plasmid p353-2 of Lactobacillus pentosus was studied for the wild-type plasmid and for a mutant plasmid with a deletion in the 5' untranslated region of the rep gene. Plasmid p353-2 codes for two countertranscript RNAs (CT-RNA) of approximately 75 and 250 nucleotides transcribed from the 5' untranslated region of the rep gene, in opposite directions. In a mutant plasmid with a deletion of the promoter and part of the CT-RNA-encoding sequence which shows a 5- to 10-fold increase in copy number compared to the wild-type plasmid, no CT-RNA could be detected. In the wild-type plasmid more than 90% of transcription initiated at a promoter upstream of the rep gene is prematurely terminated to form a 190 nucleotide truncated RNA, whereas in the mutant plasmid nearly all transcripts reach a size (1100 nucleotides) corresponding to that of the rep gene. A model is presented for the role of CT-RNA in control of plasmid replication, similar to that previously presented for the staphylococcal plasmid pT181, involving a mechanism of transcriptional attenuation of rep RNA at a site just upstream of the rep gene.

Gene disruption in Lactobacillus plantarum strain 80 by site-specific recombination: isolation of a mutant strain deficient in conjugated bile salt hydrolase activity.

A chloramphenicol-resistance gene (cml) was introduced into the Lactobacillus plantarum gene encoding conjugated bile acid hydrolase (cbh) on a ColE1 replicon. This plasmid which is nonreplicative in Lactobacillus was used to transform L. plantarum strain 80. A homologous double cross-over recombination event resulted in replacement of the chromosomal cbh gene by the cml-containing cbh gene. The transformants obtained were unable to synthesize active conjugated bile acid hydrolase (Cbh). The Cbh- CmlR phenotype was stably maintained for more than 100 generations under nonselective conditions.

Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014.

The DNA sequences of a 2.4 kb plasmid (p353-2) from Lactobacillus pentosus MD353 and a 1.9 kb plasmid (p8014-2) from Lactobacillus plantarum ATCC 8014 show 81.5% overall similarity. Both plasmids carry elements (replication protein gene, plus-origin and minus-origin of replication), which are typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR). Direct evidence for an RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. A minus-origin of replication was defined for plasmids p353-2 and p8014-2 based on DNA sequence analysis and on its ability to convert single-stranded into double-stranded plasmid DNA. Plasmids pLPE323, pLPE350 and pLPC37 that are derived from the p353-2 or p8014-2 replicon are structurally and segregationally stable in L. pentosus MD353, L. plantarum ATCC 8014 and in Lactobacillus casei ATCC 393. The presence of Escherichia coli or lambda DNA fragments in vectors derived from p353-2 or p8014-2 does not affect the structural stability but results in segregational instability of the vectors. The instability increases with increasing size of the inserted DNA fragment. Since vectors based on these replicons can be efficiently propagated in a wide variety of Lactobacillus species, they are highly suitable for cloning and expression of foreign DNA in Lactobacillus, provided that selective pressure is applied.

Organization and characterization of three genes involved in D-xylose catabolism in Lactobacillus pentosus.

A cluster of three genes involved in D-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis D-xylose isomerase (68% and 77%, respectively), and to E. coli D-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment D-xylose. NMR analysis confirmed that 13C-xylose was converted into 13C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of D-xylose isomerases of different bacteria suggests that L. pentosus D-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli D-xylose isomerase and not to a second similarity group comprising D-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5'-xylR (1167 bp, repressor) - xylA (1350 bp, D-xylose isomerase) - xylB (1506 bp, D-xylulose kinase) - 3' is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB.

Complementation of the inability of Lactobacillus strains to utilize D-xylose with D-xylose catabolism-encoding genes of Lactobacillus pentosus.

The inability of two Lactobacillus strains to ferment D-xylose was complemented by the introduction of Lactobacillus pentosus genes encoding D-xylose isomerase, D-xylulose kinase, and a D-xylose catabolism regulatory protein. This result opens the possibility of using D-xylose fermentation as a food-grade selection marker for Lactobacillus spp.

Incompatibility of Lactobacillus Vectors with Replicons Derived from Small Cryptic Lactobacillus Plasmids and Segregational Instability of the Introduced Vectors.

Three new Lactobacillus vectors based on cryptic Lactobacillus plasmids were constructed. The shuttle vector pLP3537 consists of a 2.3-kb plasmid from Lactobacillus pentosus MD353, an erythromycin resistance gene from Staphylococcus aureus plasmid pE194, and pUC19 as a replicon for Escherichia coli. The vectors pLPE317 and pLPE323, which do not contain E. coli sequences, were generated by introducing the erythromycin resistance gene of pE194 into a 1.7- and a 2.3-kb plasmid from L. pentosus MD353, respectively. These vectors and the shuttle vector pLP825 (M. Posno, R. J. Leer, J. M. M. van Rijn, B. C. Lokman, and P. H. Pouwels, p. 397-401, in A. T. Ganesan and J. A. Hoch, ed., Genetics and biotechnology of bacilli, vol. 2, 1988) could be introduced by electroporation into Lactobacillus casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis strains with similar efficiencies. Transformation efficiencies were strain dependent and varied from 10 to 10 transformants per mug of DNA. Plasmid DNA analysis of L. pentosus MD353 transformants revealed that the introduction of pLP3537 or pLPE323 was invariably accompanied by loss of the endogenous 2.3-kb plasmid. Remarkably, pLPE317 could only be introduced into an L. pentosus MD353 strain that had been previously cured of its endogenous 1.7-kb plasmid. The curing phenomena are most likely to be explained by the incompatibility of the vectors and resident plasmids. Lactobacillus vectors are generally rapidly lost when cells are cultivated in the absence of selective pressure. However, pLPE323 is stable in three of four Lactobacillus strains tested so far.

Oral administration of TNP-Lactobacillus conjugates in mice: a model for evaluation of mucosal and systemic immune responses and memory formation elicited by transformed lactobacilli.

Safe live vector systems are being developed for oral delivery of antigens. A transformation system for indigenous Lactobacillus species of the gastrointestinal tract is described. Model systems were set up to evaluate immune responses. Orally administered trinitrophenylized (TNP) Lactobacillus were examined for their ability to induce immunological memory formation via determination of specific antibody titres in serum. We demonstrate a direct correlation between the level of systemic memory formation, as revealed by specific anti-TNP IgG serum antibodies, and the TNP substitution ratio of the Lactobacillus suspension used for oral priming. The specific IgG anti-TNP serum titres were comparable to or even higher than the titres of parental intraperitoneally primed animals. These results demonstrate the feasibility of using orally administered antigen-Lactobacillus as a future approach to vaccination.

The gene for Spirodela oligorhiza chloroplast ribosomal protein homologous to E. coli ribosomal protein L16 is split by a large intron near its 5' end: structure and expression.

The nucleotide sequence of a Spirodela chloroplast DNA fragment, which directs the synthesis of a approximately 15 kD chloroplast ribosomal protein in an E. coli cell free system, has been determined. The deduced aminoacid sequence of the open reading frame shows extensive homology with E. coli ribosomal protein L16. Primer extension analysis, S1 nuclease mapping and nucleotide sequence analysis indicate that the chloroplast L16 gene (rpl16) is interrupted by a 1411 bp intron, which separates a short 5' exon from a large 3' exon. The shorter in vitro synthesized ribosomal protein results from an artificial initiation event at an internal ATG codon in the 3' exon. The sequences at the 5' and 3' splice sites of the intron are similar to consensus sequences described for other, class II intron containing, protein coding chloroplast genes. Northern hybridization experiments reveal 6 stable transcripts of rpl16 ranging from 500 b to greater than 4000 b. As determined by S1 nuclease mapping, the 3'-end of the smallest transcript maps exactly after the stem of a proposed termination signal. Finally, the implications of the finding of a cluster of several chloroplast ribosomal protein genes and possible polycistronic transcription of this chloroplast DNA region, are discussed in relation to the organization and expression of ribosomal protein genes found in the S10 operon of E. coli.

The genes encoding chloroplast ribosomal proteins S7 and S12 are located in the inverted repeat of Spirodela oligorhiza chloroplast DNA.

We have used a variety of methods to localize the genes for ribosomal proteins S7 and S12 on Spirodela chloroplast DNA. Heterologous hybridization with a rps12 gene specific probe from Euglena has revealed the presence of rps12 homologous sequences within the inverted repeat of Spirodela chloroplast DNA on the fragment BamHI-V. In the partial nucleotide sequence of this fragment, two regions of amino acid sequence homology to Euglena S12 can be identified, separated from each other by a 542 bp intron with conserved boundary sequences. As was found for Nicotiana S12, the Spirodela S12 coding regions are for 85 amino acids homologous (79%) to E. coli S12 (starting from residue 38 to the C-terminus). Likewise, we are unable to identify the 37 5' terminal codons of rps12 in Spirodela. The functionality of the Spirodela rps12 sequence is discussed. The rps7 gene is located adjacent to rps12. Chloroplast ribosomal protein C-S11 (homologous to S7) has been detected by immunoprecipitation with both a polyspecific anti 30S serum and an anti C-S11 serum, among the in vitro translation products of mRNAs selected by Spirodela chloroplast DNA fragments BamHI-V and BamHI-P. Since in a DNA dependent E. coli cell free system, only BamHI-V appears to be capable of synthesis of C-S11, it is concluded that rps7 is located entirely within BamHI-V and is transcribed into a mRNA which extends into BamHI-P. As determined by Northern hybridization experiments, rps7 is cotranscribed with rps12; a stable transcript of approx. 1100 b is detected in total cellular Spirodela RNA with either rps12 and rps7 gene specific probes. The rps12 probe additionally detects an approx. 600 b transcript, which presumably corresponds to the excised rps12 intron RNA. Finally we have examined the expression of both rps7 and rps12 during light induced chloroplast development by Northern blotting and by immunoblotting. It is shown, that the steady-state levels of neither chloroplast ribosomal protein transcripts, nor those of the chloroplast ribosomal proteins itself, change significantly during the greening process.

Regulation of protein synthesis in eukaryotes. Eukaryotic initiation factor eIF-2 and eukaryotic recycling factor eRF from neuroblastoma cells.

eIF-2 purified from neuroblastoma cells consists of three subunits, which appear to be of molecular weight identical to those of the subunits of rabbit reticulocyte eIF-2. A protein fraction has been isolated from neuroblastoma cells with characteristics similar to eRF from reticulocytes: stimulation of amino acid incorporation in a hemin-deprived reticulocyte lysate, the removal of GDP from eIF-2-GDP complexes, a 4-5-fold stimulatory effect in a two-step reaction measuring 40 S preinitiation complex formation and a 3-3.5-fold stimulation in the methionyl-puromycin synthesis. In the methionyl-puromycin-synthesizing system phosphorylated eIF-2 is not responsive to the addition of this fraction from neuroblastoma cells. The protein fraction contains eRF which seems to be similar to the eRF isolated from Ehrlich ascites tumor cells and somewhat distinct from the reticulocyte factor. Incubation of neuroblastoma cell lysate in the presence of [gamma-32P]ATP results in the phosphorylation of a protein of Mr 36 000, migrating on SDS-polyacrylamide gels to the position of eIF-2 alpha. This protein is also phosphorylated in vitro by HRI from reticulocytes. These results may reflect a common underlying principle for the quantitative regulation of protein synthesis in eukaryotic cells.

Localization of three chloroplast ribosomal protein genes at the left junction of the large single copy region and the inverted repeat of Spirodela oligorhiza chloroplast DNA.

In order to determine the localization of ribosomal protein genes on the chloroplast genome of Spirodela, we have followed two different approaches: First, antisera were prepared against purified 30S, 50S and 70S chloroplast ribosomal proteins from Spinacia. These antisera react with about two third of the chloroplast ribosomal proteins as shown by protein blot and immunoprecipitation experiments. Recombinant plasmids carrying the Spirodela BamHI-G or PstI-I cpDNA fragment both direct the synthesis of a 15 kD chloroplast ribosomal protein in a DNA dependent E. coli cell free system. This was confirmed by molecular weight determination, immunoprecipitation and competition immunoprecipitation experiments. Second, heterologous hybridization with the rps19 gene probe from Nicotiana revealed the localization of this gene on the chloroplast DNA of Spirodela within the BamHI-G fragment at the left junction of the large single copy region and the inverted repeat. Furthermore we show that the recombinant plasmid carrying Nicotiana rps19 also directs the synthesis of another chloroplast ribosomal protein in an E. coli cell free system. The identity of this protein is discussed.

Isolation, characterization, phosphorylation and site of synthesis of Spinacia chloroplast ribosomal proteins.

We have characterized the ribosomal proteins from Spinacia chloroplasts using two-dimensional gel electrophoresis. The 30S and 50S subunits contain 23-25 and 36 ribosomal proteins, respectively. In contrast to prokaryotic ribosomes, chloroplast ribosomes contain at least one (and possibly two) phosphorylated ribosomal proteins. Isolated chloroplasts synthesize in the presence of ((35)S) labeled methionine and cysteine at least seven 30S and thirteen 50S ribosomal proteins which are assembled into (pre)ribosomes. This suggests that about one third of the chloroplast ribosomal proteins is encoded by the chloroplast DNA itself. The identity of several labeled proteins in the two-dimensional gel electrophoretic patterns which did not comigrate with stained chloroplast ribosomal proteins is discussed.