Biological oxidation of arsenite: batch reactor experiments in presence of kutnahorite and chabazite.

Arsenic represents a threat to all living organisms due to its toxicity which depends on its speciation. This element is carcinogenic, teratogenic and is certainly one of the most important contaminants affecting millions of people around the world. Abiotic and biotic processes control its speciation and distribution in the environment. We have previously shown that a new bacterial strain named ULPAs1 performed oxidation of As(III) (1.33 mM) to As(V) in batch cultures. In order to develop new methods to remove arsenic from contaminated effluents or waste, by bacterial oxidation of As(III) to As(V) followed by its sorption, the conservation of oxidative properties of ULPAs1 was investigated when cultivated in batch reactors in the presence of two solid phases, chabazite and kutnahorite, already used as microorganisms immobilizing materials in biological remediation processes. In parallel, the retention efficiency of these solid phases toward arsenic ions and particularly arsenate was studied. Pure quartz sand was used as a reference material. Kutnahorite efficiently sorbed As(V), chabazite alone performed As(III) oxidation and pure quartz sand did not sorb arsenic at all. The arsenite oxidative properties of ULPAs1 were conserved when cultivated in the presence of quartz or chabazite.

pUB2380: characterization of a ColD-like resistance plasmid.

A detailed analysis of the mobilizable, ColE1-like resistance plasmid, pUB2380, is reported. The 8.5-kb genome encodes six (possibly seven) major functions: (1) a ColD-like origin of replication, oriV, with associated replication functions, RNAI and RNAII; (2) a set of active mobilization functions highly homologous to that of ColE1, including the origin of transfer, oriT; (3) a ColE1-like multimer resolution site (cer); (4) a kanamycin-resistance determinant, aph, encoding an aminoglycoside-3'-phosphotransferase type 1; (5) an insertion sequence, IS1294; and (6) two genes, probably cotranscribed, of unknown function(s). The GC content of the various parts of the genome indicates that the plasmid is a hybrid structure assembled from DNA from at least three different sources, of which the replication region, the mobilization functions, and the resistance gene are likely to have originated in the enterobacteriaceae.

IS1294, a DNA element that transposes by RC transposition.

IS1294, found on the ColD-like resistance plasmid pUB2380, is IS91-like. It is an active 1.7-kb insertion sequence that lacks terminal inverted repeats, displays insertion-site specificity, and does not generate direct repeats of the target site. The element has one large open reading frame, tnp(1294), encoding a transposase of 351 amino acids, related to members of the REP family of replication proteins used by RC-plasmids of gram-positive bacteria. IS1294 transposes using rolling-circle replication, initiated at one end of the element, oriIS, and terminated at the other, terIS. oriIS and terIS are highly conserved among like IS elements. oriIS resembles the leading strand replication origins of RC-plasmids; terIS resembles a rho-independent transcription terminator. IS1294 mediates not only its own transposition, but also sequences adjacent to terIS. A transposition model for IS1294 and related elements, involving rolling-circle replication and single-strand DNA intermediates, is presented.

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.

Extracellular transport of VirG protein in Shigella.

The ability of Shigella to spread within and between epithelial cells is a prerequisite for causing bacillary dysentery and requires the function encoded by the virG gene on the large plasmid. The outer membrane VirG (IcsA) protein is essential for bacterial spreading by eliciting polar deposition of filamentous actin (F-actin) in the cytoplasm of epithelial cells. Recent studies have indicated that an N-terminal 80-kDa VirG portion is exposed on the bacterial cell surface and released into the external medium, while the following 37-kDa C-terminal portion is embedded in the outer membrane, although little is known about the extracellular transport of the VirG protein. In this study, we attempted to elucidate the export pathway of VirG protein across the outer membrane and found that the C-terminal 37-kDa portion, termed VirG beta-core, serves as the self-transporter for the secretion of the preceding 80-kDa portion from the periplasmic side of the outer membrane to the external side. Indeed, foreign polypeptides such as MalE or PhoA covalently linked to the N terminus of VirG beta-core were transported to the external side of the outer membrane, and it was further shown that the folding structure of the passenger polypeptide at the periplasmic side of the outer membrane interferes with its translocation. Analysis of the secondary structure of VirG beta-core predicted that the critical structural property was a beta-barrel channel consisting of amphipathic anti-parallel transmembrane beta-strands, interspersed by hairpin turns and loops. These results thus strongly suggest that the secretion of VirG protein from Shigella is similar to the export system utilized by the IgA protease of Neisseria.

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.

Effects of pili rigidity and energy availability on conjugative plasmid transfer in aquatic environments.

Conjugal transfer frequencies of nonconjugative plasmid pCE325 associated with either the conjugative plasmid R388 (rigid pili) or R100-1 (flexible pili) were measured in waste water and seawater between two strains of Escherichia coli K12. These strains were selected from three strains after estimating (i) their survival capacity in the two water types and (ii) the maintenance and expression of plasmid-located genes in the different strains. Mobilization of plasmid pCE325 was always below the detection limit, but increased when organic matter was added to the microcosms. This mobilization was not related to cell growth, but to the availability of energy conditioning the physiological state of the cells. The transfer frequency was higher when the conjugative plasmid encoded flexible pili.

Why are quiescent mesophyll protoplasts from Nicotiana sylvestris able to re-enter into the cell cycle and re-initiate a mitotic activity?

Mesophyll protoplasts of Nicotiana sylvestris incubated in an adequate culture medium re-enter very rapidly into the cell cycle and divide. The transition G0/G1 is accompanied by a complete reversion of the program of gene expression. The program of the photosynthetic differentiated mesophyll cell is abolished whereas a new multipartite program of a highly stressed but ready-to-divide cell is established. Some genes encode proteins which structure suggests they may play key roles in these events. Most of the induced genes are under multiple controls: stress and/or development. Stress response and cellular re-organization might thus be closely related events that cannot be dissociated. It is probable that the re-entry of a protoplast into the cell cycle, ie the initial step of totipotency, closely depends on the coordinated activation of a set of genes that share common regulatory mechanisms.

virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the virG protein and determination of the complete coding sequence.

On the 230-kilobase-pair (kb) virulence plasmid of Shigella flexneri 2a strain YSH6000, at least seven separate genetic determinants have been identified. One of them, an approximately 4-kb region, virG, that is required for the Sereny reaction, was extensively studied to examine the role of the virG region. The phenotype of a VirG- mutant (M94) of YSH6000 in the cytoplasm of cultured MK cells was characterized by a kinetic study of the invading shigellae. The observed phenotype of M94 in the cytoplasm indicated that the virG locus is not required for multiplication of the invading shigellae, but is essential for their spread to adjacent cells. The DNA region necessary for the VirG function was localized to a 3.6-kb DNA sequence on the 230-kb plasmid. A 130-kilodalton polypeptide was confirmed to be the virG product. External labeling of bacteria with 125I indicated that the 130-kilodalton virG protein is exposed on the bacterial surface. The nucleotide sequence of 4,472 bp, which contains the functional virG gene and its own regulatory sequence, was determined, and a large open reading frame encoding 1,102 amino acid residues was identified.

Molecular genetic approaches to the pathogenesis of bacillary dysentery.

Bacillary dysentery is an invasive infectious disease of the human colon. At least three genetic loci on the chromosome and a huge plasmid have been implicated in its pathogenesis. Results obtained from molecular genetic studies, mainly of the genes, virG, virF and kcpA are discussed.

Tn3-like elements: molecular structure, evolution.

The structure and transposition mechanism of Tn3-elements are described. Different studies showed that Tn21, Tn501, Tn1721 and Tn3926 are closely related. An evolution model for these transposons is proposed.

[TTE-RAS (Titertek-Enterobac-Ras), a new micromethod for the semi-automatic identification of Enterobacteriaceae within 5 hours]. / TTE-RAS, une nouvelle microméthode pour l'identification semi-automatique des Enterobacteriaceae en 5 heures.

Titertek-Enterobac-Ras (TTE-RAS), a new semi-automated system for the identification of the Enterobacteriaceae-within five hours, has been evaluated and compared with conventional methods. This note presents the results upon 655 strains, mainly Enterobacteriaceae TTE-RAS provided correct identification for about 92 p. cent and 97 p. cent of Enterobacteriaceae respectively before and after carrying out supplementary tests according to manufacturer's instruction. TTE-RAS gave 97 p. cent specific results for Salmonella. On the other hand, the system correctly identified Aeromonas hydrophila, but not the Gram negative strict aerobic bacteria.

Antimicrobial susceptibilities of food-isolated strains of Yersinia enterocolitica, Y. intermedia, Y. frederiksenii, and Y. kristensenii.

The in vitro antimicrobial susceptibility of Yersinia enterocolitica and newly related species isolated from foods was examined. Only 4 of 375 isolates displayed resistance to non-ss-lactam antibiotics. MICs of ampicillin and carbenicillin determined by agar dilution with respect to 125 isolates showed the high susceptibility of Y. kristensenii and biovar 3 of Y. enterocolitica to carbenicillin (MIC for 90% of the strains, less than or equal to 8 micrograms/ml).

Characterization of Tn3926, a new mercury-resistance transposon from Yersinia enterocolitica.

A new transposon coding for mercury resistance (HgR), Tn3926, has been found in a strain of Yersinia enterocolitica, YE138A14. The element has a size of 7.8 kb and transposes to conjugative plasmids belonging to different incompatibility groups. A restriction map has been established. DNA-DNA hybridization indicates that Tn3926 displays homology with both Tn501 and Tn21; the greatest homology is shown with the regions of these transposons that encode HgR. Weaker homology is observed between Tn3926 sequences and those regions of Tn501 and Tn21 that encode transposition functions. Complementation experiments indicate that the Tn3926 transposase mediates transposition of Tn21, albeit somewhat inefficiently, but not of Tn501, while the resolvase mediates resolution of transposition cointegrates formed via Tn21, Tn501, or Tn1721.

Mercury resistance in Yersinia enterocolitica.

First isolation of a mercury-resistant strain of Yersinia enterocolitica is reported. This strain, named 138-A14, is resistant to mercuric chloride and merbromin, but sensitive to phenylmercuric borate and sodium merthiolate. The mercury resistance of 138-A14 is not transferable spontaneously to Escherichia coli K12 by conjugation.

Suitable conditions for characterization, identification, and isolation of the mRNA of the small subunit of ribulose-1,5-bisphosphate carboxylase from Nicotiana sylvestris.

The products synthesized in vitro by messenger RNA (mRNA) extracted from Nicotiana sylvestris were analyzed by electrophoresis on polyacrylamide slab gels. Only three of the major polypeptides synthesized are considered here: P55, P32, and P20. P55 and P32 were translated from chloroplast mRNA. P55 corresponds to the large subunit of ribulose-1,5-bisphosphate (RuP2) carboxylase; P32 is probably a chloroplast membrane protein. P20, the polypeptide synthesized from cytoplasmic poly(A)(+) RNA, is the precursor of the small subunit of RuP2 carboxylase. The balance between P20 and P32, in which their relative proportions varied inversely, was regulated by the age of the leaves and the time of illumination; we took advantage of this phenomenon to isolate the mRNA from the small subunit in relatively large amounts. This mRNA has a molecular weight of 350,000.

Comparison of proteins synthesized in vivo and in vitro by mRNA from isolated protoplasts.

Studies of proteins synthesized in vitro by messenger RNA (mRNA) extracted from tobacco protoplasts showed that the changes in protein synthesis and especially the lack of certain proteins observed previously in isolated protoplasts did not result from a failure of translation.

Changes in protein synthesis during the initial stage of life of tobacco protoplasts.

The biosynthesis of Fraction I protein in isolated protoplasts is compared with that in the plant. Radioactive precursors were incorporated into isolated protoplasts ("in vitro" labeling) and into leaves, from which the protoplasts were isolated later ("in situ" labeling). The biosynthesis of Fraction I protein stopped almost completely as soon as the protoplasts were incubated in the culture medium.