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Bacterial communities present in the rhizosphere of different plants, including rice (Oryza sativa), play an essential key role in biogeochemical cycles, plant nutrition and disease biocontrol. The Camargue area of France, part of the Rhône river delta flowing into the Mediterranean Sea, is considered a saline ecosystem. In order to understand the soil bacterial ecology of the Camargue rice growing areas, we collected samples from six different sites, from the rhizosphere of two rice varieties (Arelate and Gageron) at late stages of growth, plus adjacent bulk soil samples, during 2013 and 2014. We used pyrosequencing of PCR amplified V3-V4 regions of the 16S rRNA gene from total extracted DNA to identify the bacterial communities present, and found that the principal bacterial phyla were composed of members belonging to the Proteobacteria, Acidobacteria, Chloroflexi, Bacteroidetes and Gemmatimonadetes phyla. While the relative abundance of these phyla did not differ between the rhizosphere and bulk soil samples, the abundances varied significantly among sites and between the years of collection. The most abundant bacterial groups at both the phylum and genus levels were similar to those found in other rice growing soils, though only one halophilic genus was present in significant numbers. Our work also suggests that the challenges of rice cultivation in proximity to relatively high-salt soils can likely be overcome by current irrigation and crop-rotation strategies.

International audience; Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins known to provide protection against toxic heavy metals such as cadmium. In an attempt to increase the ability of bacterial cells to accumulate heavy metals, sheep MTII was produced in fusion with the maltose binding protein (MBP) and localized to the cytoplasmic or periplasmic compartments of Escherichia coli. For all metals tested, higher levels of bioaccumulation were measured with strains over-expressing MBP-MT in comparison with control strains. A marked bioaccumulation of Cd, As, Hg and Zn was observed in the strain over-expressing MBP-MT in the cytoplasm, whereas Cu was accumulated to higher levels when MBP-MT was over-expressed in the periplasm. Metal export systems may also play a role in this bioaccumulation. To illustrate this, we over-expressed MBP-MT in the cytoplasm of two mutant strains of E. coli affected in metal export. The first, deficient in the transporter ZntA described to export numerous divalent metal ions, showed increasing quantities of Zn, Cd, Hg and Pb being bioaccumulated. The second, strain LF20012, deficient in As export, showed that As was bioaccumulated in the form of arsenite. Furthermore, high quantities of accumulated metals, chelated by MBP-MT in the cytoplasm, conferred greater metal resistance levels to the cells in the presence of added toxic metals, such as Cd or Hg, while other metals showed toxic effects when the export systems were deficient. The strain over-expressing MBP-MT in the cytoplasm, in combination, with disruption of metal export systems, could be used to develop strategies for bioremediation.

The left end DNA of Mu-like transposable bacteriophage D3112 was sequenced from bp 2521 to bp 5483. Two large open reading frames were identified: ORF A (bp 2539-4611) and ORF B (bp 4626-5378). ORF A can encode a 690 amino acid, 78 kDa protein which is 44.4% similar to Mu transposase and ORF B can encode a 250 amino acid, 27 kDa protein, which is 46.4% similar to, though 62 amino acids shorter than, the Mu B protein. The cloned D3112 A gene exhibited activity on a mini-D3112-containing plasmid in Pseudomonas aeruginosa.

Arsenite is extremely toxic and, though non-mutagenic, is a carcinogen. To determine the effects of arsenite on changes in cell physiology, we searched for genes in HeLa cells whose mRNAs are more abundant after cellular exposure to arsenite. A cDNA subtraction was performed between cDNA synthesized from HeLa cells grown in the absence and presence of 5 microM sodium arsenite. Isolation and sequencing of three clones that showed a higher hybridization signal to RNA from arsenite-exposed cells, versus unexposed cells, revealed that two of the cDNAs coded for human ferritin H chain and the other coded for metallothionein-II. These results suggest the possibility that arsenite exposure may lead to increased levels of oxygen radicals, which augmented metallothionein and ferritin can act to detoxify.

Highly repetitive satellite DNAs comprise a significant portion of higher eukaryotic genomes and have been implicated in a variety of chromosome processes, such as centromere structure and function, that are related to their presence in heterochromatin. In addition, heterochromatin can induce metastable expression of adjacent genes. However, the role of highly repetitive satellite DNAs in these effects remains to be elucidated. In an effort to address this question, plasmids containing a human 1797-bp Eco RI satellite II DNA, plus the neo and the HSV-1 tk genes, were electroporated into a TK − /NEO − human cell line. The presence of the satellte DNA sequences within the electroporated plasmids was found to interfere with the generation of stable TK + , but not NEO + , transfectants depending on the location and/or orientation of the cloned satellite DNA.

The Escherichia coli nlp gene is highly homologous to the regulatory ner genes of transposable coliphages, Mu and D108. It was discovered, via its action when overexpressed, as a positive activator of mal gene expression in a cya − crp ∗ 1 strain. Chromosomal disruption of the nlp gene by insertion of a promoterless luxAB reporter gene revealed that nlp is nonessential for E. coli viability. Light measurements from the resulting nlp::luxAB transcriptional fusion, plus RNA dot blot analysis, suggest that nlp is transcribed. Southern-blot analyses of DNAs from several bacterial species were performed and indicate that nlp is evolutionarily conserved, but only among closely related Enterbacteriaceae.

The nucleotide sequence (2682 bp) of the left end of the Mu-like transposable bacteriophage D3112 cts15 from Pseudomonas aeruginosa was determined. A 720 bp open reading frame (ORF) is located on the bottom strand (positions 892-173), potentially encoding a polypeptide of 240 residues (Mr = 26,329). Specific binding of Escherichia coli Integration Host Factor (IHF) to a site located 907-922 bp from the D3112 left end suggests the existence of a P. aeruginosa IHF and its role, as in Mu, in the regulation of phage development.

Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed by molecular cloning. Transposable mini-Mu's with selectable markers (ampicillin resistance, kanamycin resistance or the entire lac operon of Escherichia coli) inserted between the Mu ends were also constructed. As a source of lac operon DNA, a pBR322 derivative with a 27 kb insert containing the lac operon was constructed. The plasmids with both ends of Mu (mini-Mu's) conferred full Mu immunity upon the host cells. However, the same miniMu's containing kan or lac inserts were defective in immunity. A summary of the construction and physical characterization, including restriction endonuclease cleavage maps and some of the biological properties of the plasmids, is presented

We have isolated four independent insertions of the entire 37-kb D10Scts10 genome in the low-copy-number plasmid pSC101 in vivo. They were all formed by replicative transposition during the D108 lytic cycle. The orientation of these four insertions was found to be the same, with the left ends facing towards pS101 replication, and the right end facing in the direction of all pS101 transcription, as was previously found for a Mu c ts62 insertion in pS101, pMC321. The exact sites of insertion of two of the D108 prophages, as well as the Mu prophage, have been determined by sequence analysis. All three insertions caused a 5-bp duplication of pS101 sequences at the target site, as has been found for insertions formed by conservative integration upon lysogeny. Moreover, we have determined the nucleotide sequence of the first 75 bp of the right end of D108 and, though this end is interchangeable with the right end of Mu as a substrate for either phage's transposition functions, there are a number of nucleotide differences between them.

We have cloned various amino-terminal domains of the transposable bacteriophage D108 A protein (transposase) into a high-copy-number expression vector and visualized the D108 polypeptides and fusion proteins expressed by the recombinant plasmids. By using crude protein extracts made from strains harboring these recombinant plasmids, we have performed vand competition assays and DNasel footprinting on a 32 P-end-labeled DNA restriction fragment which contained the Mu right end (to which the Mu A protein binds) and have shown that these fusion proteins in crude extracts can specifically bind to this DNA substrate. Furthermore, we have divided the amino-terminal 13 kDa of the D108 A protein (which may contain two bi-α-helical protein structures) in half and have shown that each half is capable of independent binding to the Mu att R site. These results suggest that the D108 transposase protein contains multiple DNA-binding domains which may be required for the complex protein-DNA interactions of D108 transposition.

The bacteriophage Mu ner gene has been cloned under the control of the lac UV5 promoter in the expression vector pOP95-15. The gene products of the recombinant plasmid, pUD88, visualized by in vitro coupled transcription-translation, are the bacteriophage Mu ner protein (8 kDa) and a 23 kDa protein consisting of the amino terminus of gpA (Mu transposase) fused to the carboxy terminus of β-lactamase. DNA-binding activity was measured by the retardation of migration of a 32 P-labeled DNA restriction fragment (containing the presumed ner-binding sites) in polyacrylamide gels. We have demonstrated specific association of ner to its binding sites to occur within 30 sec after the addition of impure extracts of ner overproducing cells. Much of this binding was dissociated within 30 sec by competition with a 20-fold molar excess of specific unlabeled DNA restriction fragment, but was resistant to dissociation when competed with unlabeled heterologous DNA for as long as 45 min at 37°. By adapting a method for DNA-footprinting using impure extracts of ner overproducing cells. we were able to determine that the ner-binding sites are located between nucleotides 1026 and 1058 from the Mu left end. These results support the hypothesis that ner is similar to the cro regulatory protein from bacteriophage λ and acts to regulate Mu early gene expression and the choice between lytic and lysogenic development.