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.

Characterization of Plys-proximal morphogenetic genes of transposable bacteriophage Mu.

Late during the bacteriophage Mu lytic cycle, Mu DNA must be matured and packaged from its dispersed integration sites in the host DNA in order to produce progeny virions. Whereas control of late gene transcription in Mu is becoming well understood, less is known about the phage morphogenetic process. To investigate the latter, we cloned and sequenced a approximately 4.3-kb region of the phage DNA beginning just upstream of the leftmost late promoter Plys. Previous mapping of amber mutations had located the lysis (lys) and proposed DNA maturation genes D and E in this region. When the DNA sequence was analyzed, seven potential open reading frames were found. DNA sequence analysis of amber mutations in genes D and E identified the sixth and seventh open reading frames as D and E, respectively. Cloning and expression of this region enabled production of cell-free protein extracts that specifically recognize the phage-encoded packaging sequence (pac), a characteristic exhibited by phage maturation enzymes. In addition, the E protein was found to share homology with the large subunit of many phage DNA maturation enzymes. These results support the hypothesis that D and E encode subunits of the Mu DNA maturation enzyme.

Development of a biosensor for on-line detection of tributyltin with a recombinant bioluminescent Escherichia coli strain.

A biosensor was developed for the detection of tributyltin (TBT), using a bioluminescent recombinant Escherichia coli:: luxAB strain. Dedicated devices allowed the on-line measurement of bioluminescence, pH and dissolved oxygen values and the feed-back regulation of temperature. Bacterial physiology was monitored by the measurement of the cellular density, respiratory activity and the intracellular level of ATP, glucose and acetate levels. Our results showed that a synthetic glucose medium gave a better TBT detection limit than LB medium (respectively 0.02 micro M and 1.5 micro M TBT). High growth and dilution rates ( D=0.9 h(-1)) allowed maximum light emission from the bacterium. Moreover, simple atmospheric air bubbling was sufficient to provide oxygen for growth and the bioluminescence reaction. Real-time monitoring of bioluminescence after TBT induction occurred with continuous addition of decanal up to 300 micro M, which was not toxic throughout a 7-day experiment. The design of our biosensor and the optimization of the main parameters that influence microbial activity led to the capacity for the detection of TBT.

A novel selenite- and tellurite-inducible gene in Escherichia coli.

Selenium is both an essential and a toxic trace element, and the range of concentrations between the two is extremely narrow. Although tellurium is not essential and is only rarely found in the environment, it is considered to be extremely toxic. Several hypotheses have been proposed to account for the toxic effects of selenite and tellurite. However, these potential mechanisms have yet to be fully substantiated. Through screening of an Escherichia coli luxAB transcriptional gene fusion library, we identified a clone whose luminescence increased in the presence of increasing concentrations of sodium selenite or sodium tellurite. Cloning and sequencing of the luxAB junction revealed that the fusion had occurred in a previously uncharacterized open reading frame, termed o393 or yhfC, which we have now designated gutS, for gene up-regulated by tellurite and selenite. Transcription from gutS in the presence of selenite or tellurite was confirmed by RNA dot blot analysis. In vivo expression of the GutS polypeptide, using the pET expression system, revealed a polypeptide of approximately 43 kDa, in good agreement with its predicted molecular mass. Although the function of GutS remains to be elucidated, homology searches as well as protein motif and secondary-structure analyses have provided clues which may implicate GutS in transport in response to selenite and tellurite.

Characterization of the lysogenic repressor (c) gene of the Pseudomonas aeruginosa transposable bacteriophage D3112.

Bacteriophage D3112 is a Mu-like temperate transposable phage of Pseudomonas aeruginosa. Genetic mapping and DNA sequence analysis have identified the left end of the phage genome as encoding the transposase enzyme (A) and the lysogenic (c) repressor. The c open reading frame (ORF), located at the leftmost end of the phage genome and transcribed from right to left, has four possible GTG initiation codons. Using site-directed mutagenesis, each of the four GTG codons was modified to GTA, which cannot serve as an initiation codon. Plasmids were constructed expressing either the wild-type repressor ORF or the ORFs containing the mutated GTA codons. When introduced into Pseudomonas aeruginosa, no immunity to superinfection by D3112 was observed when the second GTG had been mutated. Northern blotting analysis demonstrated that the D3112 c repressor is transcribed as a 900-nt mRNA. The promoter region was defined by transcriptional lacZ fusions and primer extension analyses to bp 972-940 from the left end of the phage genome. When the D3112 c repressor was overexpressed and purified as a fusion protein with a C-terminal six-histidine extension (cts15-His6), it showed high affinity for a 261-bp PvuII fragment localized directly upstream of the c repressor ORF. Our results indicate that although D3112 c shows higher amino acid similarity to the lambda family of repressors than it does to those of Mu and D108, it appears that its structure and function more accurately reflect an evolutionary ancestry with those from transposable coliphages Mu and D108.

The bacteriophage D108 Ner repressor binds a conformationally distinct operator.

The Ner protein encoded by the transposable coliphage D108, an 8.6 kDa lambda Cro-like repressor, binds to an operator spanning 50 bp of DNA. The distinguishing features of this operator are two perfect 11-bp inverted repeats (5'-CCGTGAGCTAC-3') that are separated by an 8-bp AT-rich spacer. Hyperreactivity of the ner operator to potassium permanganate and the hydroxyl radical indicate that the AT-rich spacer assumes a variant conformation consistent with a bend. Using an electrophoretic mobility shift assay, we demonstrated that Ner does not display significant affinity for a single 11-bp site. Furthermore, DNase I protection analysis and circular-permutation binding assays reveal that alterations in the length and sequence of the AT-rich spacer that separates the 11-bp inverted repeats significantly alter Ner-operator interactions, and demonstrate that the intrinsically bent ner operator is conformationally altered upon protein binding.

Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion.

Escherichia coli was used as a model to study initial adhesion and early biofilm development to abiotic surface. Tn10 insertion mutants of Escherichia coli K-12 W3110 were selected for altered abilities to adhere to a polystyrene surface. Seven insertion mutants that showed a decrease in adhesion harbored insertions in genes involved in lipopolysaccharide (LPS) core biosynthesis. Two insertions were located in the rfaG gene, two in the rfaP gene, and three in the galU gene. These adhesion mutants were found to exhibit a deep-rough phenotype and to be reduced, at different levels, in type 1 fimbriae production and motility. The loss of adhesion exhibited by these mutants was associated with either the affected type 1 fimbriae production and/or the dysfunctional motility. Apart from the pleiotropic effect of the mutations affecting LPS on type 1 fimbriae and flagella biosynthesis, no evidence for an involvement of the LPS itself in adhesion to polystyrene surface could be observed.

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.

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.

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.

Use of a luminescent bacterial biosensor for biomonitoring and characterization of arsenic toxicity of chromated copper arsenate (CCA).

An arsenic oxyanion-inducible Escherichia coli chromosomal operon (arsRBC) has been previously identified. Construction of a luciferase transcriptional gene fusion (arsB::luxAB) showed that ars operon expression, plus concomitant cell luminescence, was inducible in a concentration-dependent manner by arsenic salts. The present study was conducted to evaluate the potential of the arsB::luxAB transcriptional gene fusion for use as a biosensor in monitoring the toxicity of arsenic compounds. Cultures from this gene fusion strain were exposed to increasing concentrations of the wood preservative chromated copper arsenate (CCA), as well as its constituents, sodium arsenate and chromated copper solution (CC). Analysis of luciferase activity revealed that the arsB::luxAB gene fusion was expressed in response to CCA and sodium arsenate, but not to the CC solution. The detection limit of arsenic was found to be 0.01 microgram As/ml (10 parts per billion, 10 ppb) and therefore well within the range of environmental concerns. A greater induction of luminescence by arsenate was observed when cells were limited for phosphate, as phosphate can act as a competitive inhibitor of arsenate ions. Our results suggest that the E. coli arsB::luxAB fusion strain has a promising future as a specific and sensitive biosensor for monitoring bioavailable levels and toxicity of arsenic near sites where CCA-treated wood has been used.

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 beta 4, from a previous classification scheme, is renamed T1. The morphology of 36 phage species is schematically represented.

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.

Expression of the Escherichia coli chromosomal ars operon.

A chromosomally located operon (ars) of Escherichia coli has been previously shown to be functional in arsenic detoxification. DNA sequencing revealed three open reading frames homologous to the arsR, arsB, and arsC open reading frames of plasmid-based arsenic resistance operons isolated from both E. coli and staphylococcal species. To examine the outline of transcriptional regulation of the chromosomal ars operon, several transcriptional fusions, using the luciferase-encoding luxAB genes of Vibrio harveyi, were constructed. Measurement of the expression of these gene fusions demonstrated that the operon was rapidly induced by sodium arsenite and negatively regulated by the trans-acting arsR gene product. Northern blotting and primer extension analyses revealed that the chromosomal ars operon is most likely transcribed as a single mRNA of approximately 2100 nucleotides in length and processed into two smaller mRNA products in a manner similar to that found in the E. coli R773 plasmid-borne ars operon. However, transcription was found to initiate at a position that is relatively further upstream of the initiation codon of the arsR coding sequence than that determined for the E. coli R773 plasmid's ars operon.

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.

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.