The ␤-proteobacterial strain ULPAs1, isolated from an arsenic-contaminated environment, is able to efficiently oxidize arsenite [As(III)] to arsenate [As(V)]. Mutagenesis with a lacZ-based reporter transposon yielded two knockout derivatives deficient in arsenite oxidation. Sequence analysis of the DNA flanking the transposon insertions in the two mutants identified two adjacent open reading frames, named aoxA and aoxB, as well as a putative promoter upstream of the aoxA gene. Reverse transcription-PCR data indicated that these genes are organized in an operonic structure. The proteins encoded by aoxA and aoxB share 64 and 72% identity with the small Rieske subunit and the large subunit of the purified and crystallized arsenite oxidase of Alcaligenes faecalis, respectively (P. J. Ellis, T. Conrads, R. Hille, and P. Kuhn, Structure [Cambridge] 9:125-132, 2001). Importantly, almost all amino acids involved in cofactor interactions in both subunits of the A. faecalis enzyme were conserved in the corresponding sequences of strain ULPAs1. An additional Tat (twin-arginine translocation) signal peptide sequence was detected at the N terminus of the protein encoded by aoxA, strongly suggesting that the Tat pathway is involved in the translocation of the arsenite oxidase to its known periplasmic location.Arsenic is present in various environments, is released either by natural weathering of rocks or by anthropogenic sources (e.g., mining industries and agricultural practices), and is found in the oxidation states ϩ5 (arsenate), ϩ3 (arsenite), 0 (elemental arsenic), and Ϫ3 (arsine). Contamination of drinkingwater supplies with the inorganic soluble forms arsenite and arsenate has often been reported, and arsenic has been identified as a major risk for human health in different parts of the world (northeast India, Bangladesh, northwest United States) (31). The biogeochemical cycle of this element strongly depends on microbial transformation, which affects the mobility and the distribution of arsenic species in the environment (33, 41). Several bacteria involved in transformation processes comprising reduction, oxidation, and methylation of arsenic species have been described (8,11,26,36,40).The toxicological effects of arsenic are related to its chemical form and oxidation state; the organic forms are the less toxic. Among inorganic forms, As(III) is reported to be on average 100 times more toxic than the less mobile As(V) (25). Several remediation processes have been described for arsenic removal (19) based on chemical oxidation of arsenite to arsenate followed by alkaline precipitation (5, 15-17, 24). The major drawbacks of these processes are that they generate additional pollution and are expensive. This has led to the exploration of alternative methods for arsenic remediation based on its biological oxidation. Several arsenite-oxidizing bacteria have been isolated, starting with an Achromobacter strain in 1918 (14). Since then, different arsenite-oxidizing bacteria, including several Pseudomonas strains (18, 42-44), A...

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.

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 (⌬CPS-P, ⌬CPS-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.Lactobacilli are fastidious gram-positive bacteria with complex nutritional requirements resulting from numerous genetic lesions in metabolic pathways which often revert to prototrophy (24). Natural auxotrophies involved in carbamoyl phosphate (CP) biosynthesis were shown to be reversed in most cases by incubating lactobacilli in CO 2 -enriched air (2; F. Bringel, unpublished data). Metabolically, lactobacilli are at the threshold of the transition from anaerobic to aerobic life (16); Lactobacillus plantarum grows in aerobiosis but prefers microaerobiosis or increased CO 2 concentration.The arginine and pyrimidine biosynthetic pathways share CP as a common precursor. CP synthetase (CPS) catalyzes the synthesis of CP from bicarbonate, glutamine, and two molecules of ATP via a complex reaction mechanism that leads to several unstable intermediates. The X-ray crystal structure of CPS in Escherichia coli has revealed the location of three separate active sites connected by two molecular tunnels that run through the interior of the protein (33) and that would allow substrate channeling and subsequent protection of the reactive intermediates (reviewed in reference 22). Furthermore, CP biosynthesis in Pyrococcus abyssi also includes metabolic channeling, a process whereby the product of one enzyme is directly transferred to the next enzyme in the pathway without being released in the bulk solvent; CPS may interact with two CP-utilizing enzymes in the pyrimidine (aspartate carbamoyltransferase) and the arginine (anabolic ornithine carbamoyltransferase) biosynthetic pathways (29). The heterodimeric CPS enzyme is composed of a small subunit (GLN) which functions as a glutamine amidotransferase and a large synthetase subunit (SYN) that fulfills the other catalytic properties (for a re...

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Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum (M.-C. Curk, J.-C. Hubert, and F. Bringel, Int. J. Syst. Bacteriol. 46595498, 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. a-Methyla-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.Lactobacilli related to Lactobacillus plantarum are lactic acid bacteria that are important in many plant fermentations (silage, pickled vegetables, sourdough) and meat and fish fermentations (17). Some of these organisms, such as the organisms found in beer, also can be food spoilage agents (19), and some lactobacilli are also found in cavities of mammals (16).The taxonomy of L. plantarum is not satisfactory (13); this species contains strains that have similar biochemical characteristics but exhibit little similarity at the DNA level. Dellaglio et al. (9) identified the following three groups on the basis of DNA-DNA hybridization data: L. plantarum, Lactobacillus pentosus, and an atypical L. plantarum group recently reclassified as Lactobacillus paraplantarum (7). In agreement with the results of Dellaglio et al. (9), Zanoni et al. (24) revived the name L. pentosus for a distinct taxon; L. pentosus can be distinguished from L. plantarum by its ability to produce acid from D-xylose and glycerol (10,13). However, these phenotypic characteristics are not sufficient to distinguish L. plantarum from L. pentosus since some strains ferment glycerol but not D-xylose (this study) or D-xylose but not glycerol (16). Direct sequencing of 16s rRNAs by reverse transcription clarified the phylogenetic status of several groups of lactic acid bacteria but could not be used to distinguish L. plantarum from L. pentosus since the 16s ...

Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO 2 . We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum, 32 strains of L. pentosus, 15 strains of L. paraplantarum, and 10 strains of L. casei. The distribution of prototroph and auxotroph phenotypes varied between species. All L. casei strains, no L. paraplantarum strains, two L. pentosus strains, and seven L. plantarum strains required arginine for growth. Arginine auxotrophs were more frequently found in L. plantarum isolated from milk products than in L. plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In L. plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one L. pentosus strain, a high CO 2 requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated.Lactic acid bacteria (LAB) are gram-positive bacteria that have adapted to rich environments. As a result, in addition to sugars as energy and carbon sources these organisms require nucleobases, vitamins, cations, and amino acids (18). For example, some LAB associated with particular fermented foods have developed auxotrophies for specific growth factors, including orotic acid present in milk for Lactobacillus delbrueckii subsp. bulgaricus (32), a small peptide present in freshly prepared yeast extracts for the sourdough bacterium Lactobacillus sanfrancisco (1), and D-mevalonic acid for rice wine spoilage lactobacilli (33). Some authors (21) consider LAB to be a highly specialized form of life in view of their complex nutritional requirements and restricted habitats. The complex nutritional needs of LAB may be the result of two opposing evolutionary processes. A primitive LAB may have had restricted metabolism and gradually acquired new enzymatic activities. Alternatively, a chemoorganotrophic ancestor with many biochemical abilities may have evolved by progressively loosing unnecessary genes upon closer association with plants, animals, or hu...

Adsorption measurements of several actinide [thorium (Th), uranium (U)] and lanthanide [lanthanum (La), europium (Eu), ytterbium (Yb)] cations by Mycobacterium smegmatis showed that sorption kinetics followed a three-phase pattern. For 5% (w/w) bacterial suspensions at pH 1, maximum cation bios0rption per gram dry biomass corresponded to 170 ~tmol Th 4+ and 187 ~tmol UO22+ . Adsorption of all cations studied obeyed the Brunauer-Emmett-Teller isotherm, which assumes multilayer binding at constant energy. Plots for the Scatchard model showed the existence of at least two types of cation complexation site, with strong and weak affinity and negative cooperation. Th 4+ was preferentially adsorbed with respect to the other cations, although all species appeared to compete for the same sites independently of bacterial viability. Adsorption of these cations was accompanied by partial release of magnesium from the cell wall, indicating that exchange reactions occurred at magnesium (Mg)-bonding sites.

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