Genomic microbiome analyses of surface sand samples from the Kyzyl-Kum Desert (Uzbekistan): characterization and comparative study.

The Kyzyl-Kum Desert extends over an area of 300,000 Km2, in the region bordering Kazakhstan, Uzbekistan and Turkmenistan and is mainly covered by sand dunes. The Kyzyl-Kum desert is also known for its large deposits of minerals of economic interests, the exploitation of which is affecting the local ecosystem and increasing the desertification. We examined the bacterial biodiversity of surface sand samples from several sites from the Kyzyl-Kum desert using pyrosequencing of PCR amplified bacterial 16S rRNA genes from total extracted soil DNA. We also examined several physicochemical parameters of the sand samples to investigate any possible correlations between bacterial community structure and environmental drivers. The predominant bacterial phyla present in the samples were found to belong to members of the Actinobacteria, Proteobacteria and Bacteroidetes. The most abundant genera in our samples were found to belong to the Arthrobacter, Adhaeribacter and Roseomonas genera. We found that the relative abundance of members belonging to the Actinobacteria phylum, commonly found in desertic areas, increase in abundance in sites with higher content of organic matter and sulfur, whereas members of the Proteobacteria and Bacteroidetes phyla seems to diminish in abundance in coarse silt and fine-grained soils and those rich in magnesium, suggesting that those parameters might influence the bacterial community composition in this desertic area. This study is the first to provide new insights into the prokaryotic community composition from this unusual desert site.

Modification of atmospheric sand-associated bacterial communities during Asian sandstorms in China and South Korea.

The transport of desert soil into the atmosphere during desert sandstorms can affect the Earth's climate and environmental health. Asian desert sandstorms occur almost every year during the Spring, as the atmosphere in the Northern hemisphere warms. It is conceivable that these Asian desert sandstorms may transport microbes from deserts, such as the Gobi and Taklamaken deserts, over long distances in China, east Asia and the Pacific. In this study, we examined local atmospheric sand particle-associated bacterial populations collected in the absence (sterile sand exposed for 24 h to the air in the absence of a sandstorm) and presence of sandstorms in five Asian cities. We used pyrosequencing of PCR-amplified 16S rDNA genes from sand-extracted total DNA to overcome cultivation limitations of bacterial enumeration. We found that >90% of the control and sandstorm sequences could be classified as representing bacteria belonging to four phyla: Proteobacteria, Bacteriodetes, Actinobacteria and Firmicutes. The sand-associated bacterial populations in sandstorm samples were distinct from sand-associated bacteria in the absence of a sandstorm. Members of the phylum Proteobacteria were found to significantly increase in sandstorm samples (P=0.01). Principal component analyses showed that the sand-associated bacterial populations were best clustered by sampling year, rather than location. DNA sequences representing bacteria belonging to several genera (including putative human pathogens) were observed to increase in sand-associated samples from sandstorms, whereas others were found to decrease, when comparing sand-associated bacterial populations versus those in control samples, suggesting human/environmental implications of sandstorm events.

Identification and phylogeny of eukaryotic 18S rDNA phylotypes detected in chlorinated finished drinking water samples from three Parisian surface water treatment plants.

AIMS: We performed a preliminary assessment of the eukaryotic 18S rDNA diversity present in finished drinking water samples from three different surface water treatment plants supplying water to the city of Paris (France). METHODS AND RESULTS: A molecular analysis was performed on a sample from each site based on sequencing of PCR amplified and cloned 18S ribosomal RNA genes. Overall, the 18S rDNA sequences combined from all samples could be affiliated to the Amoebozoa (20.8% of the phylotypes), Ciliophora (25%), Metazoa (33.3%), Fungi (8.3%), Cercozoa (4.2%) and unclassified eukaryotes (8.3%) groups. CONCLUSIONS: The 18S rDNA sequences affiliated to the Amoebozoa, Ciliophora and Metazoa lineages were found to be the most abundant phylotypes observed in the drinking water samples. Phylotypes found to be present in two, or all three, samples (41.7% of the total) may represent groups with members adapted to drinking water treatment plant (DWTP) ecosystem conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that finished drinking water can contain 18S rDNA sequences representing a variety of eukaryotic taxa. Further research is needed to better characterize the eukaryotic biodiversity of DWTPs and the effects of the finished drinking water diversity on the downstream water distribution network.

Identification and characterization of genetically programmed responses to toxic metal exposure in Escherichia coli.

In order to identify chromosomal genetically programmed responses to toxic metal exposure, a library of 3000 Escherichia coli clones was created that contained the promoterless luxAB genes of Vibrio harveyi inserted at single and random chromosomal loci. Changes in gene expression, as measured by a change in luminescence, were monitored after exposure of the clones to various metals. In this manner, we have identified two clones that showed an increase in luminescence in the presence of aluminum, one clone in the presence of nickel, and two clones in the presence of selenite. Identification of the metal-induced gene(s), and characterization of their biochemical function, will provide important clues about the effects of these metals at the molecular level.

Characterization of the Pseudomonas aeruginosa transposable phage D3112 [corrected] left-end regulatory region.

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.

Characterization of the Pseudomonas aeruginosa transposable bacteriophage D3112 A and B genes.

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.

A cloned fragment of HeLa DNA containing consensus sequences of satellite II and III DNA hybridizes with the Drosophila P-element and with the 1.8 kb family of human KpnI fragments.

We have cloned a repetitive EcoRI fragment from the human genome which displays weak homologies with the Drosophila melanogaster transposable P-element. This cloned DNA appeared not to be a mobile element but, instead, a divergent member of human satellite II or III DNAs. We present here the first complete nucleotide sequence of a 1.797 kilobase pair (kb) satellite-like DNA. Moreover, this EcoRI satellite monomer contains a unique sequence of 49 basepairs (bp) that is devoid of the satellite consensus repeat 5'TTCCA3'. Southern hybridization analysis revealed that the cloned insert is closely related to a highly repetitive 1.8 kb KpnI family of tandemly organized satellite DNAs. Thus, the relationships among these satellite DNA families appear to be complex and may be a factor in their copy number, position and spatial organization.

The Escherichia coli Mu/D108 phage ner homologue gene (nlp) is transcribed and evolutionarily conserved among the Enterobacteriaceae.

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 Enterobacteriaceae.

Orientation and sequence analysis of right ends and target sites of bacteriophage mu and D108 insertions in the plasmid pSC101.

We have isolated four independent insertions of the entire 37-kb D108cts 10 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 pSC101 replication, and the right end facing in the direction of all pSC101 transcription, as was previously found for a Mucts62 insertion in pSC101, 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 pSC101 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.

In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers.

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 mini-Mu'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.

Cloning and localization of the repressor gene (c) of the Mu-like transposable phage D108.

We have localized the D108 thermosensitive (cts) repressor gene to a region of DNA approx. 600 base pairs (bp) in length by sub-cloning an RsaI restriction endonuclease fragment (bp 200 to bp 802 from the left-end of the D108 genome). We determined that the gene product from this fragment appears to be the same size (19 kDa) as that expressed from clones containing larger fragments of D108 DNA. Results from in vitro gel electrophoresis band-retardation and in vivo immunity assays show that the sub-cloned repressor appears to be fully functional.

Purification and characterization of the Ner repressor of bacteriophage Mu.

The Ner protein of bacteriophage Mu acts as a lambda cro-like negative regulator of the phage's early (transposase) operon. Using the band retardation assay to monitor ner-operator-specific DNA-binding activity, the 8 kDa Ner protein was purified to homogeneity. DNase I footprinting revealed that the purified protein bound and protected a specific DNA operator that contains two 12 bp sites with the consensus sequence 5'-ANPyTAPuCTAAGT-3', separated by a 6 bp spacer region. Moreover, regions corresponding to a turn of the DNA helix flanking these 12 bp repeats are also protected by Ner. Unlike the functionally similar lambda cro protein, gel filtration experiments show that the native molecular mass of Mu Ner to be approx. 8 kDa. These results, plus the pattern of DNase I protection, suggest that the protein may bind as a monomer to each of its specific DNA substrates.

The bacteriophage Mu transposase protein can form high-affinity protein-DNA complexes with the ends of transposable elements of the Tn 3 family.

The 37 kb transposable bacteriophage Mu genome encodes a transposase protein which can recognize and bind to a consensus sequence repeated three times at each extremity of its genome. A subset of this consensus sequence (5'-PuCGAAA(A)-3') is found in the ends of many class II prokaryotic transposable elements. These elements, like phage Mu, cause 5 bp duplications at the site of element insertion, and transpose by a cointegrate mechanism. Using the band retardation assay, we have found that crude protein extracts containing overexpressed Mu transposase can form high-affinity protein-DNA complexes with Mu att R and the ends of the class II elements Tn 3 (right) and IS101. No significant protein-DNA complex formation was observed with DNA fragments containing the right end of the element IS102, or a non-specific pBR322 fragment of similar size. These results suggest that the Mu transposase protein can specifically recognize the ends of other class II transposable elements and that these elements may be evolutionarily related.

A bacterial toxicity assay performed with microplates, microluminometry and Microtox reagent.

We have developed a procedure for undertaking a Microtox-based test by coupling microplate and microluminometric technologies. Sample dilutions are prepared in a 96-well polystyrene microplate kept at 15 degrees C, while the Microtox reagent and diluent are placed in an opaque, microluminometry-compatible 96-well microplate also kept at 15 degrees C. Exposure begins when sample aliquots are brought into contact with bacterial reagents in the opaque microplate. After specific exposure times (5, 15, 30 and 60 min), bacterial luminescence is rapidly measured by placing the opaque microplate in a microluminometer. Reproducibility of the procedure, as well as general agreement of EC50 end-point values with published reports, is demonstrated herein after toxicity trials with six metals (Ni2+, Cd2+, Zn2+, Pb2+, Cu2+ and Cr6+). Results suggest that a 60-min exposure time may have value in getting more "sensitivity mileage" out of this Microtox-based assay. This microplate procedure possesses attractive features that augment sample throughput and information output. Further refinement and validation studies are ongoing in our laboratories.

Mu transposase-stimulated illegitimate recombination of Tn3kan- and IS101-containing plasmids.

The transposable bacteriophage Mu and the mobile genetic elements Tn3 and IS101 replicatively transpose to random target sites, produce 5 bp target site duplications, and contain the sequence 5'-PuCGAAAPu-3' starting at bp 21 from their ends. The presence of these shared characteristics, plus the fact that Mu transposase can specifically bind to the termini of Tn3 and IS101 in vitro, suggests that the elements may be evolutionarily conserved and retain some functional capacity to transpose each other's DNA. To examine this proposition, in vivo transposition-mating assays were performed and demonstrated that Mu transposase stimulated the formation of recA-independent recombination products between Tn3kan- or IS101-containing plasmids and a target plasmid (pOX38cam) up to 200-fold. However, when transferred to recA+ hosts, these recA-independent products yielded resolution products suggestive of illegitimate recombination, as similar recombination and resolution products were generated, at reduced frequencies, in the absence of Mu transposase. Thus, Mu transposase may stimulate a host-mediated, recA-independent illegitimate recombination reaction. As adjacent pSC101 sequences, including a formerly unknown but functional IHF site (bp 2238-2251), were required for Mu transposase-stimulated IS101 illegitimate recombination, IHF may be one of the putative host factors involved in these recombination reactions.

The detection and characterization of genetically programmed responses to environmental stress.

The rapid, accurate, and inexpensive detection of environmental contaminants (and the stress that they engender) is still a major problem worldwide. Though assays exist for monitoring these pollutants, they can often be expensive, time-consuming, and require extensive equipment and/or training in order to be effective. Research over the past decade has pointed to the measurement of enzymes encoded by genes programmed to respond to particular classes of environmental stress as a means of quantifying altered environmental health. The detection of physical and chemical contaminants can thus be performed using standard enzyme assays, by measuring the quantity of these enzymes (e.g. via immunoassays), or through the use of the technology of "gene-fusions." In this latter case, the genes encoding easily quantified enzymes are "fused" (cloned) such that their expression is under the control of genes whose expression is induced in the presence of these contaminants. In these cases, the measurement of the reporter gene's activity from the sample would signal the presence of a particular chemical and/or physical contaminant. The advantages of this system are its rapidity, ease of use, and single end-point measurement, thus allowing a "one box" (single detector) solution to measurements of environmental quality and health. Moreover, these systems can be designed for on-line monitoring and computer-aided operation for use in a wide variety of settings.

Identification of Tn10 insertions in the dsbA gene affecting Escherichia coli biofilm formation.

Escherichia coli was used as model to study initial adhesion and early biofilm development to an abiotic surface. Tn10 insertion mutants with reduced attachment to a polystyrene surface were isolated. Three adhesion mutants harbored the transposon in the dsbA gene, whose product, DsbA, catalyses folding of numerous extracytoplasmic disulfide bond-containing proteins. All three mutants were weakly adherent and grew poorly. Cell surface structure analysis showed that motility. type 1 fimbriation and lipopolysaccharide structure were affected in these mutants. The pleiotropic effect of the dsbA mutations on biofilm formation is discussed.

Bioluminescence-based assays for detection and characterization of bacteria and chemicals in clinical laboratories.

OBJECTIVES: To survey recent advances in the application of bioluminescence to public health problems. The usefulness of bacterial (lux) and eucaryotic (luc) luciferase genes is presented, along with several examples that demonstrate their value as "reporters" of many endpoints of clinical concern. CONCLUSIONS: The development of new technologies for monitoring biological and chemical contaminants is in continuous progress. Recent excitement in this area has come from the use of genes encoding enzymes for bioluminescence as reporter systems. Applications of the recombinant luciferase reporter phage concept now provide a sensitive approach for bacterial detection, their viability, and sensitivity to antimicrobial agents. Moreover, a number of fusions of the lux and luc genes to stress inducible genes in different bacteria can allow a real-time measurement of gene expression and determination of cellular viability, and also constitute a new tool to detect toxic chemicals and their bioavailability.

Expression of domains of mouse alpha-fetoprotein in Escherichia coli.

A mouse alpha-fetoprotein complementary DNA containing the coding region for amino acids 256 to 548 was fused to the lac transcriptional and translational control elements contained on the expression vector pOP203-28. The expression of a 35 kD hybrid lac Z'- alpha-fetoprotein polypeptide in Escherichia coli was demonstrated by the chloramphenicol release assay and by immunoprecipitation using rabbit anti-mouse alpha-fetoprotein antibodies as probe.

Taxonomic changes in tailed phages of enterobacteria.

Out of 136 new phages, 80 (59%) are classified into 23 species according to morphology and physicochemical properties. Six new species are described and species b4, from a previous classification scheme, is renamed T1. The morphology of 36 phage species is schematically represented.