In Lactobacillus plantarum, carbamoyl phosphate is synthesized by two carbamoyl-phosphate synthetases (CPS): carbon dioxide differentiates the arginine-repressed from the pyrimidine-regulated CPS.

Carbamoyl phosphate (CP) is an intermediate in pyrimidine and arginine biosynthesis. Carbamoyl-phosphate synthetase (CPS) contains a small amidotransferase subunit (GLN) that hydrolyzes glutamine and transfers ammonia to the large synthetase subunit (SYN), where CP biosynthesis occurs in the presence of ATP and CO(2). Lactobacillus plantarum, a lactic acid bacterium, harbors a pyrimidine-inhibited CPS (CPS-P; Elagöz et al., Gene 182:37-43, 1996) and an arginine-repressed CPS (CPS-A). Sequencing has shown that CPS-A is encoded by carA (GLN) and carB (SYN). Transcriptional studies have demonstrated that carB is transcribed both monocistronically and in the carAB arginine-repressed operon. CP biosynthesis in L. plantarum was studied with three mutants (DeltaCPS-P, DeltaCPS-A, and double deletion). In the absence of both CPSs, auxotrophy for pyrimidines and arginine was observed. CPS-P produced enough CP for both pathways. In CO(2)-enriched air but not in ordinary air, CPS-A provided CP only for arginine biosynthesis. Therefore, the uracil sensitivity observed in prototrophic wild-type L. plantarum without CO(2) enrichment may be due to the low affinity of CPS-A for its substrate CO(2) or to regulation of the CP pool by the cellular CO(2)/bicarbonate level.

Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment.

Arsenic is ubiquitous in the biosphere and frequently reported to be an environmental pollutant. Global cycling of arsenic is affected by microorganisms. This paper describes a new bacterial strain which is able to efficiently oxidize arsenite (As[III]) into arsenate (As[V]) in liquid medium. The rate of the transformation depends on the cell density. Arsenic species were separated by high performance liquid chromatography (HPLC) and quantified by inductively coupled plasma-atomic emission spectrometry (ICP-AES). The strain also exhibits high minimum inhibitory concentrations (MICs) for As[III] (6.65 mM (500 mg L-1)) and other heavy metals, such as cadmium (1.42 mM (160 mg L-1)) or lead (1.20 mM (250 mg L-1)). Partial identification of the strain revealed a chemoorganotrophic, Gram-negative and motile rod. The results presented here demonstrate that this strain could represent a good candidate for arsenic remediation in heavily polluted sites.

Identification of the plasmid-mobilization potential of the strain Klebsiella pneumoniae ozenae KIIIA isolated from a polluted aquatic environment.

The Klebsiella pneumoniae ozenae KIIIA strain was isolated from the River Rhine soon after a serious mercury pollution episode and was selected for mercury resistance as well as for intergeneric DNA mobilization helper potential. This transfer helper capacity was shown to be related to the presence of a Tn3-like transposable element, Tn5403. Because transposon-mediated fusion was found to be involved in the mobilization potential of KIIIA, the visualization and the identification of the conjugative element, responsible for the transfer, were necessary. Our results show that, in addition to the four nonconjugative plasmids visualized in a previous study, K. pneumoniae ozenae KIIIA harbors two other plasmids, pK130 and pK45, of respective sizes of 130 and 45 kb, but none of these plasmids is involved in the mobilization mechanism. The presence of yet another extrachromosomal element pK225, with a size of 225 kb, was established by indirect methods, since yields of pK225 isolated from KIIIA were low and the plasmid was difficult to visualize directly. However, the integration of this plasmid into the chromosome was not detected. The present paper highlights the problem of detecting some plasmids in bacteria which have been isolated from the environment. For these plasmids, indirect approaches, that detect conjugative functions, constitute a feasible alternative for the investigation of the plasmid content of bacteria, if the direct approach fails. An analysis of the different types of transconjugants indicated that the mercury-resistance marker as well as the mobilization potentials, expressed by KIIIA, are linked to pK225. This plasmid could not be assigned to a described Inc group either by DNA hybridization or by PCR amplification.

Recombinant plasmid mobilization between E. coli strains in seven sterile microcosms.

Transfer by mobilization of a pBR derivative recombinant plasmid lacking transfer functions (oriT+, tra-, mob-) from one E. coli K12 strain to another was investigated in seven sterile microcosms corresponding to different environments. These microcosms were chosen as representative of environments that genetically engineered microorganisms (GEMOs) encounter after accidental release, namely attached biomass in aquatic environments (biofilm), soil, seawater, freshwater, wastewater, mouse gut, and mussel gut, GEMOs survived in the same way as the host strains in all microcosms. Recombinant DNA mobilization occurred in the mouse gut, in sterile soil, and in biofilm. The plasmid transfer rates principally reflected the environmental conditions encountered in each microcosm.

Arginine biosynthesis and regulation in Lactobacillus plantarum: the carA gene and the argCJBDF cluster are divergently transcribed.

A cluster of citrulline biosynthetic genes has been cloned and sequenced from a fragment of Lactobacillus plantarum CCM 1904 (ATCC 8014) DNA isolated as complementing a Bacillus subtilis argF mutation. The gene order was carA-argCJBDF, with carA transcribed divergently from the arg cluster. Although other gram-positive bacteria show similar arg clusters, this arrangement for carA is thus far unprecedented. Downstream from the arg cluster, two open reading frames (ORF7 and ORF8) having unknown functions were found. Sequence analysis of the end of a 10.5-kb cloned DNA fragment showed that argF was 3.5 kb from the ldhL gene coding for L-(+)-lactate dehydrogenase. A tree representation of amino acid sequence clustering relationships of 31 ornithine carbamoyltransferases (OTCases) from various organisms revealed two prokaryotic groups: one with ArgF of L. plantarum and one with ArgF of B. subtilis, which are paralogous. This divergence was not observed in vivo because an L. plantarum argF mutant (AM 1215) harboring no OTCase activity was complemented by the argF genes of L. plantarum and B. subtilis. No OTCase activity was detectable when L. plantarum was grown in the presence of saturating amounts of arginine or citrulline. Arginine may repress the citrulline biosynthetic genes in L. plantarum by using 11 identified DNA motifs which resemble the Escherichia coli ARG box consensus and which are in most cases separated by multiples of 11 bp, corresponding to a DNA helical turn. The carA and argCJBDF genes are divergently transcribed. Their putative promoters are 6 bp apart and are partially overlapped by putative ARG boxes, suggesting concerted transcription regulation.

Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese.

Among 1,962 bacterial isolates from a smear-surface soft cheese (Munster cheese) screened for activity against Listeria monocytogenes, six produced antilisterial compounds other than organic acids. The bacterial strain WHE 92, which displayed the strongest antilisterial effect, was identified at the DNA level as Lactobacillus plantarum. The proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action of the antilisterial compound produced by this bacterium suggested that it was a bacteriocin. Purification to homogeneity and sequencing of this bacteriocin showed that it was a 4.6-kDa, 44-amino-acid peptide, the primary structure of which was identical to that of pediocin AcH produced by different Pediococcus acidilactici strains. We report the first case of the same bacteriocin appearing naturally with bacteria of different genera. Whereas the production of pediocin AcH from P. acidilactici H was considerably reduced when the final pH of the medium exceeded 5.0, no reduction in the production of pediocin AcH from L. plantarum WHE 92 was observed when the pH of the medium was up to 6.0. This fact is important from an industrial angle. As the pH of dairy products is often higher than 5.0, L. plantarum WHE 92, which develops particularly well in cheeses, could constitute an effective means of biological combat against L. monocytogenes in this type of foodstuff.

Structure and organisation of the pyrimidine biosynthesis pathway genes in Lactobacillus plantarum: a PCR strategy for sequencing without cloning.

This report describes the sequence and structural organisation of the pyrimidine biosynthesis pathway genes of Lactobacillus plantarum CCM 1904. It also describes an in vitro technique based on PCR for sequencing without cloning. This new technique was developed because it was impossible to clone certain parts of the L. plantarum genomic DNA in the Escherichia coli host. L. plantarum pyr genes are organised as a 9.8-kb operon with the following order: pyrR, pyrB, pyrC, pyrAA, pyrAB, pyrD, pyrF and pyrE. There are two major differences from the pyrimidine operons of Bacillus subtilis (Quinn et al., J. Bacteriol. 266 (1991) 9113-9127; Turner et al., J. Bacteriol, 176 (1994) 3708-3722) and Bacillus caldolyticus (Ghim et al., Microbiology 140 (1994) 479-491): the absence of pyrP encoding for uracil permease, and the absence of an open reading frame named orf2, whose function is unknown. Two mutually exclusive stem-loop structures were predicted at the 5'-end of L. plantarum pyr mRNA; this operon could be regulated by transcriptional attenuation under the control of PyrR. Complementation of E. coli pyrD, pyrF and pyrE mutants was obtained with a L. plantarum genomic DNA library. Alignment of the L. plantarum Pyr proteins with other known procaryotic Pyr proteins indicates that they display highly conserved regions in Gram-positive and Gram-negative bacteria.

Characterization of lactobacilli by Southern-type hybridization with a Lactobacillus plantarum pyrDFE probe.

Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum (M.-C. Curk, J.-C. Hubert, and F. Bringel, Int. J. Syst. Bacteriol. 46:595-598, 1996) can hardly be distinguished on the basis of their phenotypes. Unlike L. plantarum and L. paraplantarum, L. pentosus ferments glycerol and xylose but not melezitose. We identified two L. pentosus strains (CNRZ 1538 and CNRZ 1544) which ferment glycerol and melezitose but not xylose. alpha-Methyl-D-mannoside was fermented by 66% of the L. plantarum strains tested but not by L. paraplantarum strains. In this paper we describe a simple method to identify L. plantarum, L. pentosus, and L. paraplantarum. This method is based on nonradioactive Southern-type hybridization between BglI DNA digests of the lactobacilli tested and a DNA probe (L. plantarum pyrDFE genes from strain CCM 1904). A total of 68 lactobacilli were classified into five groups on the basis of the bands detected. Two groups contained L. plantarum strains; one of these groups contained 31 strains, including the type strain, and was characterized by bands at 7, 4, and 1 kb, and the other group contained strain LP 85-2 and was characterized by bands at 5 and 1.1 kb. Only one band (a band at around 7 kb) was detected in the strains belonging to the L. pentosus group, and two bands (at 4 and 1 kb) were found in the strains belonging to the L. paraplantarum group. No hybridization was detected in the last group, which contained Lactobacillus casei, Lactobacillus coryniformis, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus delbrueckii, and Lactobacillus leichmannii strains.

Lactobacillus paraplantarum sp. now., a new species related to Lactobacillus plantarum.

Four strains of facultatively heterofermentative lactobacilli isolated from beer and human feces have physiological characteristics similar to those of Lactobacillus plantarum. Unlike 66% of the L. plantarum strains tested (F. Bringel, M.-C. Curk, and J.-C. Hubert, Int. J. Syst. Bacteriol. 46:588-594, 1996), these strains do not catabolize alpha-methyl-D-mannoside. However, because they exhibit little DNA relatedness to L. plantarum and Lactobacillus pentosus, these four strains were classified as members of a new species, Lactobacillus paraplantarum; strain CNRZ 1885 (= CIP 104668) is the type strain.

Identification of a new transposon Tn5403 in a Klebsiella pneumoniae strain isolated from a polluted aquatic environment.

A Klebsiella pneumoniae strain having mobilization "helper" potential has been isolated from the river Rhine. Analysis of the transconjugants resulting from the mobilization of non-conjugative pBR-type plasmids and RSF1010 derivatives showed that the transfer-helper capacity of the K. pneumoniae strain is related to the presence of a Tn3-like transposable element, Tn5403. This element has been identified and localized in a plasmid.

Determination of chromosome size and number of rrn loci in Lactobacillus plantarum by pulsed-field gel electrophoresis.

The size of the Lactobacillus plantarum CCM 1904 chromosome was determined by pulse-field gel electrophoresis. It was found to be 3.3-3.4 Mb using SfiI or AscI restriction endonucleases, compared to 3-4 Mb found for the other L. plantarum strains tested. L. plantarum CCM 1904 5S rDNA was clonedl by polymerase chain reaction, sequenced, and used as a probe to characterize strains. At least five rrn loci were found. The pulsed-field gel electrophoresis macrorestriction patterns were strain-specific, while the rDNA restriction hybridization patterns were species-specific.

Complexes of mycobactin from Mycobacterium smegmatis with scandium, yttrium and lanthanum.

The interaction of cations of group IIIb elements (Sc, Y, La) with mycobactin S in ethanol leads to the formation of 1:1 complexes which closely resemble the known aluminium compound with respect to ultraviolet absorption and fluorescence emission spectra. Determination of molar stoichiometry by spectrophotometry shows that this method can be conveniently applied to the estimation of purity in mycobactin samples. Hydrolytic dissociation measurements based on aqueous extraction of the labelled complexes in heterogeneous phase indicate a pronounced gradation in cation-binding stability, which increases from La (rapid and complete dissociation) to sc (approximately 24% dissociation under similar conditions). The observed properties of the complexes are rationalized by semi-empirical model calculations, which suggest that ionic radius effects resulting from interaction of the IIIb cations with mycobactin S would not favour octahedral coordination of these elements as in the stable Fe(III) complex.

Studies on transcription of the yeast URA2 gene.

The multifunctional protein carbamoylphosphate synthetase (CPSase)-aspartate transcarbamylase (ATCase) encoded by the URA2 gene catalyses the first two steps of the yeast pyrimidine pathway. An excess of the final product, the intracellular UTP (uridine triphosphate), inhibits both the transcription of the URA2 gene and the enzymatic activities. Results presented in this paper suggest that transcription of URA2 is negatively regulated (repression-derepression) and establish that this regulation is less efficient in the flow of the pyrimidine pathway than feedback inhibition.

Cloning and structure of the pyrE gene of Lactobacillus plantarum CCM 1904.

The pyrE gene of Lactobacillus plantarum CCM 1904, coding for the orotate phosphoribosyl transferase involved in the pyrimidine biosynthetic pathway, was cloned in Escherichia coli and sequenced. The predicted polypeptide sequence extending over 212 amino acids (MW 22,690) was compared to those of E. coli and to those of lower eukaryotes (Saccharomyces cerevisiae, Podospora anserina, Sordaria macrospora, Dictyostelium discoideum). Important conserved stretches were revealed, implying that these proteins are closely related.

Cloning and sequencing of URA10, a second gene encoding orotate phosphoribosyl transferase in Saccharomyces cerevisiae.

Orotate phosphoribosyl transferase (OP-RTase) catalyses the transformation of orotate to OMP in the pyrimidine pathway. In the yeast Saccharomyces cerevisiae, the URA5 gene is known to encode this enzyme activity. In this paper we present the cloning and sequencing of a yeast gene, named URA10, encoding a second OPRTase enzyme. Comparison of the predicted amino acid sequences between URA5 and URA10 genes shows more than 75% similarity. These sequences have also been compared to those of Escherichia coli, Podospora anserina, Sordaria macrospora and Dictyostelium discoideum. Remarkable similarities in the primary structure of these proteins have been found. Gene disruption experiments revealed that URA10 gene expression is responsible for the leaky phenotype of a ura5 mutant. Assays of OPRTase activity in extracts from ura5 and ura10 mutants indicate that the URA10 product contributes only 20% of the total activity found in wild type cells.

Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction.

A small 2.1-kb plasmid called pLP1 was extracted from Lactobacillus plantarum CCM 1904 (ATCC 8014) and cloned into the Escherichia coli pUC19 plasmid. As determined by DNA-DNA Southern hybridization with a pLP1-radioactively labeled probe, other lactic acid bacteria such as L. curvatus, L. sake, Carnobacterium, and Leuconostoc mesenteroides harbor pLP1-related plasmids. Shuttle vectors based on the pLP1 replicon were constructed by inserting the erythromycin-resistance gene from pVA891 into the various pUC19-pLP1 constructions. pLP1-based shuttle vector transformation efficiencies (TE) by electroporation were compared to TE of a broad-host-range plasmid pGK12 in different lactobacilli strains. Expression of the pUC19-pLP1 plasmids in Escherichia coli maxicells showed that pLP1 encodes for a 37,000 MW protein which can act in trans allowing the replication of plasmids in which this protein is truncated. The pLP1-based shuttle vectors producing this protein replicate in lactobacilli and also in Bacillus subtilis. A pLP1-free strain was obtained by incompatibility with a pLP1-based shuttle vector introduced in L. plantarum CCM 1904 by electroporation. The absence of pLP1 has no incidence on the strain phenotype suggesting that pLP1 is not essential for the strain in our laboratory conditions.

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904.

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.

Cloning and analysis of the gene for the major outer membrane lipoprotein from Pseudomonas aeruginosa.

The gene for the Pseudomonas aeruginosa outer membrane lipoprotein I was isolated from a genomic library in the phage lambda EMBL3 vector and subsequently subcloned in the low copy-number, wide host-range plasmid vector, pKT240. The cloned gene was highly expressed, resulting in the production of a low molecular-weight protein (8 kD) that was found to be associated with the outer membrane. Sequence analysis showed an open reading frame of 83 amino acids with a putative N-terminal hydrophobic signal peptide of 19 residues immediately followed by the lipoprotein consensus sequence, GLY-CYS-SER-SER (residues 19-22). The predicted amino acid composition of the mature polypeptide and that of the purified lipoprotein I of P. aeruginosa (Mizuno and Kageyama, 1979) were identical. In contrast with other Gram-negative outer membrane lipoproteins, conformation predictions suggested that the mature protein was a single alpha helix.

Structure and expression of the URA5 gene of Saccharomyces cerevisiae.

The URA5 gene of Saccharomyces cerevisiae encodes orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase) which catalyses the transformation of orotate to OMP in the pyrimidine pathway. We present in this paper the cloning and the sequencing of this gene, the last in the yeast pyrimidine pathway to be cloned. We have deduced the protein sequence of the OPRTase of S. cerevisiae from the DNA sequence and compared it to that of Escherichia coli, Podospora anserina and Dictyostelium discoideum. Some important similarities in the structure of these four proteins have been found. Finally, we have quantified the transcription of the URA5 gene in different physiological conditions and confirmed that it was not under the control of UTP or any intermediary product of the pathway.