Relationships between arginine degradation, pH and survival in Lactobacillus sakei.

Lactobacillus sakei is one of the most important lactic acid bacteria of meat and fermented meat products. It is able to degrade arginine with ammonia and ATP production by the arginine deiminase pathway (ADI). This pathway is composed of three enzymes: arginine deiminase, ornithine transcarbamoylase and carbamate kinase, and an arginine transport system. The transcription of the ADI pathway is induced by arginine and subjected to catabolite repression. In order to understand the physiological role of the degradation of this amino acid we investigated the growth of bacteria under various conditions. We show that arginine degradation is responsible for an enhanced viability during the stationary phase when cells are grown under anaerobiosis. Arginine is necessary for the induction of the ADI pathway but in association with another environmental signal. Using a mutant of the L-lactate dehydrogenase unable to lower the pH we could clearly demonstrate that (i) low pH is not responsible for cell death during the stationary phase, so survival is due to another factor than elevated pH, (ii) neither low pH nor oxygen limitation is responsible for the induction of the ADI pathway together with arginine since the ldhL mutant is able to degrade arginine under aerobiosis.

Molecular cloning and analysis of the ptsHI operon in Lactobacillus sake.

The ptsH and ptsI genes of Lactobacillus sake, encoding the general enzymes of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), were cloned and sequenced. HPr (88 amino acids), encoded by ptsH, and enzyme I (574 amino acids), encoded by ptsI, are homologous to the corresponding known enzymes of other bacteria. Nucleotide sequence and mRNA analysis showed that the two genes are cotranscribed in a large transcript encoding both HPr and enzyme I. The transcription of ptsHI was shown to be independent of the carbon source. Four ptsI mutants were constructed by single-crossover recombination. For all mutants, growth on PTS carbohydrates was abolished. Surprisingly, the growth rates of mutants on ribose and arabinose, two carbohydrates which are not transported by the PTS, were accelerated. This unexpected phenotype suggests that the PTS negatively controls ribose and arabinose utilization in L. sake by a mechanism different from the regulation involving HPr described for other gram-positive bacteria.

Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes.

Single-crossover homologous integration in Lactobacillus sake was studied. Integration was conducted with nonreplicative delivery vector pRV300. This vector is composed of a pBluescript SK- replicon for propagation in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to integrate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequency was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 x 10(3) integrants/micrograms of DNA. We used this integration strategy to inactivate the ptsI gene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system, and the lacL gene, which is one of the two genes required for the synthesis of a functional beta-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake.

Carbohydrate Utilization in Lactobacillus sake.

The ability of Lactobacillus sake to use various carbon sources was investigated. For this purpose we developed a chemically defined medium allowing growth of L. sake and some related lactobacilli. This medium was used to determine growth rates on various carbohydrates and some nutritional requirements of L. sake. Mutants resistant to 2-deoxy-d-glucose (a nonmetabolizable glucose analog) were isolated. One mutant unable to grow on mannose and one mutant deficient in growth on mannose, fructose, and sucrose were studied by determining growth characteristics and carbohydrate uptake and phosphorylation rates. We show here that sucrose, fructose, mannose, N-acetylglucosamine, and glucose are transported and phosphorylated by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS permease specific for mannose, enzyme II(supMan), was shown to be responsible for mannose, glucose, and N-acetylglucosamine transport. A second, non-PTS system, which was responsible for glucose transport, was demonstrated. Subsequent glucose metabolism involved an ATP-dependent phosphorylation. Ribose and gluconate were transported by PTS-independent permeases.

Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome.

To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of approximately 4 kb. The replicon was derived from a broad-host-range plasmid, pAM beta 1, which replicates by a unidirectional theta mechanism and its thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and approximately 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.

Developmental potentials of enteric neural crest-derived cells in clonal and mass cultures.

The aim of this work was to test the differentiation capacities of neural crest-derived cells that had already migrated to the gut and were in the process of forming the intrinsic gut plexuses, when they were withdrawn from the gut wall environment. The experiments were performed on the quail bowel of embryos at 4 to 12 days of incubation (E4 to E12), which was dissociated into a single cell suspension. The crest cells invading the gut carry the NC1/HNK1 epitope. This allowed us to select them as singlets under the control of a uv-illuminated inverted microscope. Crest-derived cells from the gizzard were then clonally cultured on a feeder layer of growth-inhibited 3T3 fibroblasts, as described before for cephalic and truncal neural crest cells. The clonal efficiency of these cells reached about 28% from E4 to E7 and decreased sharply at E8. The size of the clones generated by cells taken between E4 to E8 varied considerably. Large clones (> 10(3)) were numerous from E4 and E5 gizzard-derived cells, whereas E7 and E8 cells produced only very small colonies. The phenotypic diversity of the clones decreased similarly during the time period under scrutiny. Two phenotypes never encountered in the enteric plexuses were found in the cultures: adrenergic cells and glial cells expressing SMP, a marker exclusively expressed by Schwann cells in vivo and normally absent in enteric glia. The capacity to yield adrenergic cells was present up to E6 in clonal cultures of gizzard neural crest-derived cells. Mass cultures of cells dissociated from the gizzard and bowel, including gut mesenchymal and epithelial cells in addition to cells of neural crest origin, revealed that both adrenergic and SMP+ cells could arise under these conditions even in a defined culture medium devoid of serum and chick embryo extract. The number of TH+ cells developing in bowel cultures reached a peak at E7, while at this stage no TH+ cells could be obtained from gizzard cultures. A craniocaudal gradient of disappearance of adrenergic precursors in the gut could thus be demonstrated. Finally we point out that these and previous experiments from other laboratories show that precursor cells of the sympathoadrenal lineage are present in all types of avian sensory and autonomic ganglia during development even though, in most of them, these cells never express the adrenergic phenotype.

The ontogeny of the neural crest.

The neural crest is part of a larger embryonic structure, the neural folds, belonging to the neural primordium of the Vertebrate embryo. The neural fold is formed by the anterior and lateral ridges of the neural anlage, which fuse mediodorsally when the neural tube closes. Anteriorly, the epithelium of the neural fold does not convert into mesenchymal cells and yields Rathke's pouch, the olfactory organ and the epithelium of the mouth roof, of the upper lip and of the frontal region of the head. From the level of the diencephalon (at the level of the epiphysis) downwards the neural fold epithelium undergoes the epitheliomesenchymal transition and yields the neural crest cells which become later on highly diversified and form various structures and tissues throughout the body. A large amount of data have shown that the environmental cues exerted on crest cells both during their migration and when they have reached their target sites are critical in determining their fate. In order to understand the mechanisms through which environmental factors influence crest cell differentiation, the developmental capacities of single neural crest cells were investigated at different time points of their ontogeny. Single cell cultures of crest cells have revealed that already at the migratory stage the neural crest is made up of cells at different states of determination. In particular, the analysis of clones obtained from single cell cultures of cephalic migratory crest cells has shown that, although many clonogenic cells are multipotent to varying degrees, others are committed to give rise to one single derivative. Totipotent progenitors able to generate representatives of virtually all the phenotypes (neuronal, glial, melanocytic and mesectodermal) encountered in cephalic neural crest derivatives were also found. We proposed that they represent stem cells analogous to those which in the hemopoietic system generate the various types of blood cells. The neural crest stem cell gives rise to diverse progenitors that become progressively restricted in their potentialities according to an essentially stochastic mechanism while dividing during and after completion of the migration process. Similar cloning experiments of crest cells that have already reached their target organs, i. e. sensory ganglia or enteric plexuses, showed that the phenotypic repertoire expressed by crest-derived cells decreases with increasing embryonic age. Efforts are made to elucidate the nature of the factors which influence either the survival and/or the differentiation of neural crest cells in the various types of environments in which they evolve.(ABSTRACT TRUNCATED AT 400 WORDS)

Both forms of translational initiation factor IF2 (alpha and beta) are required for maximal growth of Escherichia coli. Evidence for two translational initiation codons for IF2 beta.

The gene infB codes for two forms of translational initiation factor IF2; IF2 alpha (97,300 Da) and IF2 beta (79,700 Da). IF2 beta arises from an independent translational event on a GUG codon located 471 bases downstream from IF2 alpha start codon. By site-directed mutagenesis we constructed six different mutations of this GUG codon. In all cases, IF2 beta synthesis was variably affected by the mutations but not abolished. We show that the residual expression of IF2 beta results from translational initiation on an AUG codon located 21 bases downstream from the mutated GUG. Furthermore, two forms of IF2 beta have been separated by fast protein liquid chromatography and the determination of their N-terminal sequences indicated that they resulted from two internal initiation events, one occurring on the previously identified GUG start codon, the other on the AUG codon immediately downstream. We conclude that two forms of IF2 beta exist in the cell, which differ by seven aminoacid residues at their N terminus. Only by mutating both IF2 beta start codons could we construct plasmids that express only IF2 alpha. A plasmid expressing only IF2 beta was obtained by deletion of the proximal region of the infB gene. Using a strain that carries a null mutation in the chromosomal copy of infB and a functional copy of the same gene on a thermosensitive lysogenic lambda phage, we could cure the lambda phage when the plasmids expressing only one form of IF2 were supplied in trans. We found that each one of the two forms of IF2, at near physiological levels, can support growth of Escherichia coli, but that growth is retarded at 37 degrees C. This result shows that both forms of IF2 are required for maximal growth of the cell and suggests that they have acquired some specialized but not essential function.

Induction of DNA amplification in the Bacillus subtilis chromosome.

A system allowing the induction of DNA amplification in Bacillus subtilis was developed, based on a thermosensitive plasmid, pE194, stably integrated in the bacterial chromosome. An amplification unit, comprising an antibiotic resistance marker flanked by directly repeated sequences, was placed next to the integrated plasmid. Activation of pE194 replication led to DNA amplification. Two different amplification processes appeared to take place: one increased the copy number of all sequences in the vicinity of the integrated plasmid and was possibly of the onion skin type, while the other increased the copy number of the amplification unit only and generated long arrays of amplification units. These arrays were purified and shown to consist mainly of directly repeated amplification units but to also contain non-linear regions, such as replication forks and recombination intermediates. They were attached to the chromosome at one end only, and were, in general, not stably inherited, which suggests that they are early amplification intermediates. Longer arrays were detected before the shorter ones during amplification. When the parental amplification unit contained repeats which differed by a restriction site the arrays which derived thereof contained in a majority of cases only a single type of repeat. We propose that the amplified DNA is generated by rolling circle replication, and that such a process might underlie a number of amplification events.

Developmental potentialities of cells derived from the truncal neural crest in clonal cultures.

The developmental potentialities of single truncal neural crest derived cells were analysed in clonal cultures. The clone-forming ability and differentiation potential of crest cells migrating through the somitic mesoderm of 3-day-old embryos (E3) and of non-neuronal cells of dorsal root ganglia taken at E6-14 were compared. Since most of the cells present in the sclerotomal and rostral parts of the somite at E3 become later on incorporated into the spinal ganglia, one can consider that these two cell populations represent the same derivatives of the trunk neural crest at different developmental stages. After 10 days in vitro, the size of clones and their phenotypic composition varied noticeably, revealing a certain heterogeneity in the founder cell populations in terms of developmental potencies. Clones obtained from migrating neural crest cells at E3 were often large (greater than 1000 cells) and many of them contained neuronal and non-neuronal cells. Dorsal root ganglion cells produced mostly small clones (less than 100 cells) in which only non-neuronal (i.e. glial) phenotypes were expressed. Therefore, both the capacity for proliferation and the differentiation ability of cloned neural crest derived cells decrease considerably with increasing embryonic age. This is even more striking if these results are compared with those obtained previously in our laboratory with single cells cultures of E2 cephalic neural crest. In the latter case, both clone sizes and cellular diversity within the colonies were much higher than with E3 truncal crest and dorsal root ganglia (DRG) non-neuronal cells. The second result of the present work concerns the differentiation of the dormant autonomic neuronal precursors of the DRG. It has been established previously that the non-neuronal cells of the DRG include adrenergic precursors than can differentiate in mass culture of dissociated DRG cells. We show that these cells never differentiate in clonal cultures but depend upon the cell density of the culture. This suggests that cell to cell interaction between crest derived cells are critical in eliciting the differentiation of the adrenergic phenotype.

Characterization of the translational start site for IF2 beta, a short form of Escherichia coli initiation factor IF2.

The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (alpha and beta) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigorous determination of the nature of the initiation triplet for IF2 beta, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2 alpha and IF2 beta. Three types of experiments were carried out. First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2 alpha synthesis. Under these conditions, IF2 beta is still formed. Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ. Any synthesis of a fused protein with beta-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2 beta initiation site within an 18-base region containing an in-phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2 alpha start sequences but containing genetic information for this 18-base region. A hybrid protein with beta-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2 beta, demonstrating that GUG, located 471 bases downstream from the IF2 alpha external start codon, is the internal start codon for the shorter form of IF2.

An eIF-4A-like protein is a suppressor of an Escherichia coli mutant defective in 50S ribosomal subunit assembly.

The assembly of ribosomes in bacterial cells is a complex process that remains poorly characterized. The in vitro assembly of active ribosomal subunits from purified RNA and protein components indicates that all of the information for proper assembly resides in the primary sequences of these macromolecules. On the other hand, the in vitro requirement of unphysiological heating steps suggests that this pathway may not accurately reflect the in vivo pathway, and that other proteins may be required. One approach to identify any additional proteins is to isolate second-site revertants of mutants defective in ribosome assembly. Ribosomal protein L24 is essential in the assembly of 50S subunits. We have identified an Escherichia coli gene, srmB, that, when expressed at high copy number, can suppress the effect of a temperature-sensitive lethal mutation in L24. The SrmB amino-acid sequence has sequence identity with mouse translation initiation factor eIF-4A and with the human nuclear protein, p68. The purified SrmB protein is a nucleic acid-dependent ATPase, like eIF-4A, but can also bind RNA in the absence of ATP and other auxiliary protein factors. The RNA dependent ATPase activity of SrmB suggests that like, eIF-4A, it could be involved in specific alterations of RNA secondary structure.

Promoter vectors with restriction-site banks.

New vectors harboring the promoter for the chloramphenicol acetyl transferase gene (cat promoter) have been constructed. These vectors are all derived from pJRD184 [Heusterspreute et al., Gene 39 (1985) 299-304], which contains a restriction-site bank. The cat promoter has been inserted at various positions and in reverse orientations so that almost all the restriction sites originally present on JRD184 can be used in cloning experiments. The expression of the aceK gene of Escherichia coli cloned under the control of the cat promoter has been tested. A large increase in the synthesis of the isocitrate dehydrogenase kinase, the aceK gene product, has demonstrated the efficiency of the newly constructed vectors.

The protein synthesis initiation factor 2 G-domain. Study of a functionally active C-terminal 65-kilodalton fragment of IF2 from Escherichia coli.

Protein synthesis initiation factor 2 (IF2) is present in Escherichia coli cells as two forms which are expressed from the same gene: IF2 alpha [97.3 kilodaltons (kDa)] and IF2 beta (79.7 kDa). During isolation, a smaller form, IF2 gamma, is generated, presumably by partial proteolysis. It has been purified to homogeneity and has an apparent mass of 70 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectrophoresis of IF2 alpha and IF2 gamma shows that IF2 gamma is immunologically partially identical with IF2 alpha. The sequence of the 15 N-terminal amino acid residues of IF2 gamma was determined and compared with that of IF2 alpha. The N-terminal amino acid of IF2 gamma corresponds to Arg-290 of IF2 alpha, suggesting that IF2 gamma is generated by proteolytic cleavage of the Lys-289-Arg-290 bond of IF2. Assuming a C terminus identical with IF2 alpha, we calculate that IF2 gamma comprises 601 amino acid residues and has a mass of 64.8 kDa. The truncated protein was tested for activities characteristic of IF2 in three in vitro assays: fMet-tRNA(fMet) binding to 70S ribosomes, N-terminal dipeptide synthesis in a DNA-dependent transcription/translation system, and ribosome-dependent GTP hydroly97-7. The specific activities of IF2 gamma were comparable with, or only slightly less than, those for IF2 alpha, indicating that IF2 gamma contains the active centers for interaction with fMet-tRNA(fMet), ribosomes, and GTP. A central region in the primary structure of IF2 shows extensive sequence homology with a number of GDP-binding proteins and especially with the G-domain of elongation factor Tu (EF-Tu).(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro expression of the Escherichia coli nusA-infB operon.

The expression of the nusA-infB operon has been investigated using an in vitro system based on the formation of the first dipeptide of the gene product. A series of plasmids containing various deletions of the operon were used as templates in this study. Of the four genes coding for protein products, 15Ka, nusA, infB, and 15Kb, only 15Ka was not expressed in this dipeptide system. The initial dipeptides for the other gene products, fMet-Asn (pnusA), fMet-Thr (IF-2 alpha), and fMet-Ala (p15Kb), were synthesized even from plasmids lacking the primary promoters. It appears that secondary (internal) promoters in the operon can efficiently direct the expression of these genes. No regulation of the expression was observed with IF-2 alpha, but pnusA inhibited the expression of the nusA gene (autoregulation) as well as the p15b gene. Experiments using an uncoupled system indicated that the effect of pnusA on nusA expression was at the level of transcription, but that both a transcriptional and a post-transcriptional effect of pnusA was seen on 15Kb expression.

Two translational initiation sites in the infB gene are used to express initiation factor IF2 alpha and IF2 beta in Escherichia coli.

The gene infB codes for the two forms of translational initiation factor IF2: IF2 alpha (97 300 daltons) and IF2 beta (79 700 daltons). To determine whether the two forms differ at their N terminus, purified IF2 alpha and IF2 beta were subjected to 11 or more steps of Edman degradation. The N-terminal amino acid sequences are completely different, but match perfectly the DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 bp downstream. A fusion was constructed between the proximal half of the infB gene and the lacZ gene lacking the region coding for the first eight amino acids. The fused gene expresses two products of 170 000 and 150 000 daltons, corresponding to the fused proteins IF2 alpha-beta-galactosidase and IF2 beta-beta-galactosidase, which confirms in vivo that the IF2 forms differ at their N terminus. A deletion of the 5'-non-translated region of the fused gene, including the Shine/Dalgarno ribosomal binding site, results in the expression of IF2 beta-beta-galactosidase but not IF2 alpha-beta-galactosidase. This strongly suggests that IF2 beta results from independent translation rather than from a precise proteolytic cleavage of IF2 alpha. Further evidence for initiation of protein synthesis at the putative IF2 alpha and IF2 beta start sites was sought by using an in vitro dipeptide synthesis assay. A DNA fragment containing the entire infB gene was cloned into three plasmid vectors and the resulting recombinant DNAs were used as templates in assays containing fMet-tRNA and various labelled aminoacyl-tRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)