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Desert-like areas located in the eastern portion of the state of Utah (USA) have geographic features that can resemble the surface of the planet Mars, characterized by red-colored hills, soils and sandstones. We examined the bacterial biodiversity of surface soil samples from several sites from the Colorado Plateau Desert in eastern Utah using pyrosequencing of PCR amplified bacterial 16S rRNA genes from total extracted soil DNA. The sample sites cover the Great Basin, Goblin Valley State Park and nearby regions on the Colorado Plateau. We also examined several physicochemical parameters of the soil 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 Proteobacteria, Actinobacteria, Bacteroidetes, and Gemmatimonadetes. The most abundant genera in our samples were found to belong to the Cesiribacter, Lysobacter, Adhaeribacter, Microvirga and Pontibacter genera. We found that the relative abundance of Proteobacteria and Gemmatimonadetes were significantly correlated with soil pH and a low concentration of organic matter, suggesting that, in these relatively high-altitude desert soils, these two parameters may be of primary importance to influence bacterial community composition.

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.

The Desert of Maine, not a real desert, is a 160,000 m2 tourist attraction of glacial silt which resembles a desert, surrounded by a pine forest in the state of Maine located in the northeastern USA. Though not a true desert, the soil of the Desert of Maine has a sandy texture with poor water-holding abilities, nutrient retention capabilities, and a relatively low pH value (pH 5.09). Samples from this site may be of interest to examine the bacterial diversity present on mineral sandy loam soils with an acidic pH, low concentrations of organic materials though surrounded by a pine forest, and compare it with true desert soil microbial populations. Two surface sand samples from the Desert of Maine were obtained, and pyrosequencing of PCR amplified 16S rRNA genes from total extracted DNA was used to assess bacterial diversity, community structure, and the relative abundance of major bacterial taxa. We found that the soil samples from the Desert of Maine displayed high levels of bacterial diversity, with a predominance of members belonging to the Proteobacteria and Actinobacteria phyla. Bacteria from the most abundant genus, Acidiphilium, represent 12.5% of the total 16S rDNA sequences. In total, 1394 OTUs were observed in the two samples, with 668 OTUs being observed in both samples. By comparing Desert of Maine bacterial populations with studies on similar soil environments, we found that the samples contained less Acidobacteria than soils from acid soil forests, and less Firmicutes plus more Proteobacteria than oligotrophic desert soils. Interestingly, our samples were found to be highly similar in their composition to an oak forest soil in France.

International audience; Salinity is an important environmental factor influencing microbial community composition. To better understand this influence, we determined the bacterial communities present in 17 different sites of brackish sediment (underwater) and soil (surface) samples from the Camargue region (Rhone river delta) in southern France during the fall of 2013 and 2014 using pyrosequencing of the V3-V4 regions of the 16S rRNA genes amplified by PCR. This region is known for abundant flora and fauna and, though saline, 30% of rice consumed in France is grown here. We found that bacterial abundance in 1g of soil or sediment, calculated by qPCR, was higher in sediments than in surface soil samples. Members belonging to the Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes phyla dominated the bacterial communities of sediment samples, while members belonging to the Proteobacteria, Bacteroidetes, Gemmatimonadetes, Actinobacteria, Firmicutes and Acidobacteria phyla dominated the bacterial communities of the soil samples. The most abundant bacterial genera present in the saline sediments and soils from the Camargue belonged mostly to halophilic and sulphate reducing bacteria, suggesting that the Camargue may be a valuable system to investigate saline, yet agriculturally productive, sediment and soil microbial ecosystem.

Enterococcus faecalis is an opportunistic pathogen that has emerged as a major cause of nosocomial infections worldwide. Many clinical strains are indeed resistant to last resort antibiotics and there is consequently a reawakening of interest in exploiting virulent phages to combat them. However, little is still known about phage receptors and phage resistance mechanisms in enterococci. We made use of a prophageless derivative of the well-known clinical strain E. faecalis V583 to isolate a virulent phage belonging to the Picovirinae subfamily and to the P68 genus that we named Idefix. Interestingly, most isolates of E. faecalis tested&mdash, including V583&mdash, were resistant to this phage and we investigated more deeply into phage resistance mechanisms. We found that E. faecalis V583 prophage 6 was particularly efficient in resisting Idefix infection thanks to a new abortive infection (Abi) mechanism, which we designated Abi&alpha, It corresponded to the Pfam domain family with unknown function DUF4393 and conferred a typical Abi phenotype by causing a premature lysis of infected E. faecalis. The abi&alpha, gene is widespread among prophages of enterococci and other Gram-positive bacteria. Furthermore, we identified two genes involved in the synthesis of the side chains of the surface rhamnopolysaccharide that are important for Idefix adsorption. Interestingly, mutants in these genes arose at a frequency of ~10&minus, 4 resistant mutants per generation, conferring a supplemental bacterial line of defense against Idefix.

Recent research suggests that corals including cold-water corals harbor a diverse community of bacteria that are not only pathogens but also potential mutualists. Here we review data on bacterial community composition and diversity on the main cold-water corals framework builder species: Lophelia pertusa and Madrepora oculata. Sampling strategies such as box core, video grabs and remotely operated vehicle did not reveal strong differences between bacterial community composition as long as samples were used that looked ‘not contaminated’. However, there were strong differences of bacterial diversity between the two coral species. An analysis of bacterial community composition by pyrosequencing of L. pertusa and M. oculata revealed for the Mediterranean Sea the presence of the potential mutualists already found in the Atlantic indicating a species-specific core microbiome. The data also suggest some biogeographical differences between the Mediterranean Sea and the North Atlantic for both coral species, however, this depends on the phylogenetic levels applied. In addition, there was also indication for a shared microbiome between the Mediterranean Sea and the Atlantic. Therefore species-specific bacterial associations seem to exist, whereas the biogeographical variability can be seen as adaptation to specific environmental conditions.

To better understand microbial populations present in atypical soil environments, and to discern any relations between these environments and their bacterial communities, we examined the “Padza de Dapani” on the island of Mayotte off the east coast of Africa. This area is not a true (hot) desert, but resembles one in many places due to extensive soil erosion. We collected surface soil samples from five different sites of the Padza de Dapani in Mayotte. We examined bacterial biodiversity using pyrosequencing of PCR-amplified 16S V1–V3 rDNA sequences from total extracted DNA. Our results show that in the acidic (pH 4.6–6), oligotrophic (organic carbon; 0.1–0.7 g/kg of soil) and mineralized (Fe: 18 g/100 g; Al: 12 g/100 g) Dapani Padza soil samples, members of the Actinobacteria and Proteobacteria phyla dominated the bacterial communities, as was also observed in samples from Saudi Arabia hot desert sands.Interestingly, members belonging to the genera Acinetobacter, Arthrobacter and Bacillus were also found to be very abundant in our samples. These were also seen in hot Asian deserts sand samples, such as those from the Gobi (Mongolia) and Taklamaken (China) deserts, thus possibly pointing to microbial populations characteristic of denuded soils.

Arid regions represent nearly 30 % of the Earth's terrestrial surface, but their microbial biodiversity is not yet well characterized. The surface sands of deserts, a subset of arid regions, are generally subjected to large temperature fluctuations plus high UV light exposure and are low in organic matter. We examined surface sand samples from the Taklamaken (China, three samples) and Gobi (Mongolia, two samples) deserts, using pyrosequencing of PCR-amplified 16S V1/V2 rDNA sequences from total extracted DNA in order to gain an assessment of the bacterial population diversity. In total, 4,088 OTUs (using ≥97 % sequence similarity levels), with Chao1 estimates varying from 1,172 to 2,425 OTUs per sample, were discernable. These could be grouped into 102 families belonging to 15 phyla, with OTUs belonging to the Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria phyla being the most abundant. The bacterial population composition was statistically different among the samples, though members from 30 genera were found to be common among the five samples. An increase in phylotype numbers with increasing C/N ratio was noted, suggesting a possible role in the bacterial richness of these desert sand environments. Our results imply an unexpectedly large bacterial diversity residing in the harsh environment of these two Asian deserts, worthy of further investigation.

International audience; Members of the pathogenic Legionella genus encounter suitable growth conditions in nuclear power plant cooling circuits. To limit its proliferation and ensure that levels remain below regulatory thresholds, chemical treatment with monochloramine can be used in continuous or sequential conditions. The aim of this study was to determine the impact of monochloramine on L. pneumophila subpopulations in the cooling circuits of a nuclear power plant. The chosen procedure involved monitoring the diversity and dynamics of L. pneumophila subpopulations every month over the course of a year in a nuclear power plant cooling circuit, which was treated for 2 months during the period under study. This study confirmed the effectiveness of monochloramine to limit L. pneumophila concentrations in cooling circuits. The culturable L. pneumophila community was strongly affected by the injection of monochloramine. Several subpopulations persisted during treatment at low concentrations (below the detection limit of standard methods), suggesting that the susceptibility of L. pneumophila is strain dependent. Although the composition of the subpopulations was not similar, the resilience of the community structure was observed. Indeed, the community eventually returned to its initial structure and presented a similar pattern of richness, diversity and uniformity to that seen before treatment.

Members of the Legionella genus find suitable conditions for their growth and survival in nuclear power plant cooling circuits. To limit the proliferation of Legionella pathogenic bacteria in nuclear power plant cooling circuits, and ensure that levels remain below regulatory thresholds, monochloramine treatment can be used. Although the treatment is highly effective, i.e. it reduces Legionella numbers by over 99%, Legionella bacteria can still be detected at low concentrations and rapid re-colonisation of circuits can occur after the treatment has ceased. The aim of this study was to develop an in vitro methodology for determining the intrinsic susceptibility of L. pneumophila strains, collected from various nuclear power plant cooling circuits subjected to different treatment conditions. The methodology was developed by using an original approach based on response surface methodology (RSM) combined with a multifactorial experimental design. The susceptibility was evaluated by the Ct factor. The susceptibility of environmental strains varies widely and is, for some strains, greater than that of known tolerant species; however, strain susceptibility was not related to treatment conditions. Selection pressure induced by monochloramine use did not result in the selection of more tolerant Legionella strains and did not explain the detection of Legionella during treatment or the rapid re-colonisation of cooling circuits after disinfection has ceased.

International audience; Life in arid regions and, in particular, hot deserts is often limited due to their harsh environmental conditions, such as large temperature fluctuations and low amounts of water. These extreme environments can influence the microbial community present on the surface sands and any rhizosphere members surrounding desert plant roots. The Jizan desert area, located in Saudi Arabia, supports particular vegetation that grows in the large sandy flat terrain. We examined five different samples, four from the rhizosphere of pioneer plants plus a surface sand sample, and used pyrosequencing of PCR-amplified V1-V3 regions of 16S rDNA genes from total extracted DNA to reveal and compare the bacterial population diversity of the samples. The results showed a total of 3,530 OTUs in the five samples, calculated using ≥ 97% sequence similarity levels. The Chao1 estimation of the bacterial diversity fluctuated from 637 to 2,026 OTUs for a given sample. The most abundant members found in the samples belong to the Bacteroidetes, Firmicutes and Proteobacteria phyla. This work shows that the Jizan desert area of Saudi Arabia can contain a diverse bacterial community on the sand and surrounding the roots of pioneer desert plants. It also shows that desert sand microbiomes can vary depending on conditions, with broad implications for sandstone monument bacterial communities

Bacteriophages are the most abundant life forms in the biosphere. They play important roles in bacterial ecology, evolution, adaptation to new environments, and pathogenesis of human bacterial infections. Here, we report the complete genomic sequences, and predicted proteins of 27 bacteriophages of the Gram-positive bacterium Staphylococcus aureus . Comparative nucleotide and protein sequence analysis indicates that these phages are a remarkable source of untapped genetic diversity, encoding 2,170 predicted protein-encoding ORFs, of which 1,402 cannot be annotated for structure or function, and 522 are proteins with no similarity to other phage or bacterial sequences. Based on their genome size, organization of their gene map and comparative nucleotide and protein sequence analysis, the S. aureus phages can be organized into three groups. Comparison of their gene maps reveals extensive genome mosaicism, hinting to a large reservoir of unidentified S. aureus phage genes. Among the phages in the largest size class (178–214 kbp) that we characterized is phage Twort, the first discovered bacteriophage (responsible for the Twort-D'Herelle effect). These phage genomes offer an exciting opportunity to discern molecular mechanisms of phage evolution and diversity.

Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases.

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.

Contamination of the environment by toxic compounds is a problem of global concern and in addressing this problem, it is necessary to identify the mechanisms by which specific agents exert their toxic effects, and develop effective, inexpensive strategies for detecting compounds in their biologically active and available form. We have used reporter gene fusion technology to identify genes, in the genetically well-characterized bacterium Escherichia coli, whose expression is affected by specific environmental toxins. As an added benefit of our approach, we have elaborated methods to use these gene fusion clones as biosensors to detect specific toxic agents, such as arsenic oxyanions. Arsenic is an abundant and useful element which is also an environmental toxin that can pose severe risks to health. Arsenic toxicity varies with oxidation state, organometallic form, and bioavailability. The Escherichia coli arsB fusion strains respond specifically to arsenic in its toxic, oxyanionic form, and can detect bioavailable amounts of these oxyanions in contaminated water samples. Combinations of different biosensor clones and assay automation will augment the use of luminescent biosensors for the detection of specific toxic agents in the environment.

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.

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 increase of arsenic (As) concentration on the earth’s surface is due both to natural sources, such as volcanic activity and weathering processes, and to anthropogenic sources, such as mining activities, agricultural and forestry applications. One example of anthropogenic arsenic contamination in the environment is the use of arsenic-containing wood preservatives. The most extensively used wood preservative is chromated copper arsenate (CCA), which is pressurised into the wood through a process called ‘Wolmanizing’ [1]. Wood intended for marine uses receives 24–40 kg CCA per cubic meter of wood to prevent its destruction by bacteria, fungi and insects. Each of the three chemicals in CCA is known to be toxic to aquatic biota at concentrations above trace levels, and found to be leached from the treated wood in both fresh and sea water [2–4]. Chemicals leached from CCA-treated wood can affect organisms that grow on the wood itself and those that live adjacent to the CCA-treated bulkheads, and also be adsorbed onto sediments, where they can be slowly released or taken up by benthic organisms [4–6]. The rate of metal accumulation in sediments and in benthos differs with each chemical in the order: Cu>As>Cr, and decreases with distance and time [1, 7]. Benthic organisms living near CCA-treated bulkheads were found to contain elevated levels of Cu and As. The number of individuals, as well as the species diversity, were also decreased at sites adjacent to CCA-treated bulkheads [1]. Pathologic and genotoxic effects have also been observed in oysters (Crassostrea virginica) living on CCA-treated wood [8]. In addition, CCA was shown to affect the growth of PCP-degrading bacterial species and their ability to degrade PCP [9, 10]. Thus, monitoring of bioavailable amounts of CCA released by the treated wood, is important in order to detect and rectify its toxic effects. Among the three chemical constituents of CCA, arsenic is the most abundant in the environment, and known to have carcinogenic and teratogenic effects on humans upon chronic exposure [11, 12]. Therefore, the focus of this study is on biomonitoring of arsenic toxicity.

International audience; Arid zones cover over 30 % of the Earth's continental surface. In order to better understand the role of microbes in this type of harsh environment, we isolated and characterized the bacteriophages from samples of the surface sand of the Mesquite Flats region via electron microscopy and DNA sequencing of a select number of cloned phage DNAs. An electron microscopic analysis of the recovered virus-like particles revealed at least 11 apparently different morphotypes sharing structural characteristics of the Caudoviridae family of tailed phages. We found that 36 % of the sequences contained no significant identity (e-value >10(-3)) with sequences in the databases. Pilot sequencing of cloned 16S rRNA genes identified Bacteroidetes and Proteobacteria as the major bacterial groups present in this severe environment. The majority of the 16S rDNA sequences from the total (uncultured) bacterial population displayed ≤96 % identity to 16S rRNA genes in the database, suggesting an unexplored bacterial population likely adapted to a desert environment. In addition, we also isolated and identified 38 cultivable bacterial strains, the majority of which belonged to the genus Bacillus. Mitomycin-C treatment of the cultivable bacteria demonstrated that the vast majority (84 %) contained at least one SOS-inducible prophage.

International audience; Legionella bacteria are ubiquitous in aquatic environments. Members of the species Legionella pneumophila are responsible for more than 98% of cases of Legionnaires' disease in France. Our objective was to validate a molecular typing method called infrequent restriction site PCR (IRS PCR), applied to the study of the ecology of Legionella and to compare this method with reference typing methods, pulsed-field gel electrophoresis (PFGE) and sequence-based Typing (SBT). PFGE and SBT are considered as gold methods for the epidemiological typing of Leg. pneumophila strains. However, these methods are not suitable to an ecological monitoring of Legionella in natural environments where a large number of strains has to be typed. Validation of IRS PCR method was performed by the identification of 45 Leg. pneumophila isolates from cooling circuits of thermal power plants by IRS PCR, PFGE and SBT. The parameters of each method were measured and compared to evaluate the effectiveness of IRS PCR. The results of this study showed that IRS PCR has a discriminating power similar or better than that of the reference methods and thus that, by its speed and low cost represents an appropriate tool for the study of the ecology of Legionella in cooling circuits.

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.

International audience; Ocean acidification may stimulate primary production through increased availability of inorganic carbon in the photic zone, which may in turn change the biogenic flux of dissolved organic carbon (DOC) and the growth potential of heterotrophic bacteria. To investigate the effects of ocean acidification on marine bacterial assemblages, a two-by-three factorial mescosom experiment was conducted using surface sea water from the East Greenland Current in Fram Strait. Pyrosequencing of the V1-V2 region of bacterial 16S ribosomal RNA genes was used to investigate differences in the endpoint (Day 9) composition of bacterial assemblages in mineral nutrient-replete mesocosms amended with glucose (0 μm, 5.3 μm and 15.9 μm) under ambient (250 μatm) or acidified (400 μatm) partial pressures of CO(2) (pCO(2) ). All mesocosms showed low richness and diversity by Chao1 estimator and Shannon index, respectively, with general dominance by Gammaproteobacteria and Flavobacteria. Nonmetric multidimensional scaling analysis and two-way analysis of variance of the Jaccard dissimilarity matrix (97% similarity cut-off) demonstrated that the significant community shift between 0 μm and 15.9 μm glucose addition at 250 μatm pCO(2) was eliminated at 400 μatm pCO(2) . These results suggest that the response potential of marine bacteria to DOC input may be altered under acidified conditions.

7 pages; International audience; We have isolated a novel Siphoviridae phage (named Sol-P11) morphotype from the surface sands of the Sahara Desert with a ribbon-like structure at the tail extremity. Sol-P11 was found to grow on a Bacillus subtilis strain isolated from the same environment and to contain a double stranded DNA genome of approximately 120 kb in length incapable of being hydrolysed by a wide variety of restriction endonucleases. The major constituent proteins of CsCl-purified Sol-P11 virions were 65, 50, 30, and 24 kDa in size, with the 30 kDa polypeptide being the major protein of the 85 nm diameter icosahedral capsid, and the other three proteins comprising the major polypeptides of the tail (320 nm in length) and ribbon-like structure. Moreover, different sized phages displaying a Sol-P11 morphology were observed in phage preparations from the Death Valley and Namib deserts. Sol-P11-like phage morphotypes have been previously described, including PBPI, a flagellum-specific phage that infects B. pumilis and phage BcP15 infecting the marine bacterium, Burkholderia cepacia DR11. We thus propose that Sol-P11 represents a member of a novel morphotype of Siphoviridae phages that use a ribbon-like structure, instead of caudal fibers, to attach to their host cell.

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.

Temperate bacteriophage Mu's 37 kilobase pair (kb) linear, double-stranded DNA requires DNA sequences at both termini in cis to transpose in a duplicative manner and thus propagate its genome during the lytic cycle. To define any spatial or orientation parameters between these sequences, we constructed "isomer" plasmids pMD186 and pMD861 containing identical Mu termini in close (or inverse) orientation 407 base pairs apart, or separated by 9.3 kb of plasmid pSC 101 DNA, respectively. Both mini-Mu plasmids transposed at equal frequencies onto an F′ episome in vivo, in the presence of an induced helper Mu prophage. However, while the vast majority of pMD861 mini-Mu transposition and maturation events were found to be the result of duplicative transposition (cointegrates), virtually all pMD186 transpositions yielded simple linear insertions of the mini-Mu plasmid without concomitant duplication of the mini-Mu. However, the construction of pMD186 mini-Mu plasmid derivatives, containing increased lengths of DNA sequences cloned between the ends of the Mu genome, yielded increasing frequencies of duplicative transposition products. These results suggest that the distance between the Mu termini can affect the final outcome of the transposition reaction.Key words: DNA transposition, phage Mu, cointegrates.

Bacteriophage Mu DNA, like other transposable elements, requires DNA sequences at both extremities to transpose. It has been previously demonstrated that the transposition activity of various transposons can be influenced by sequences outside their ends. We have found that alterations in the neighboring plasmid sequences near the right extremity of a Mini-Mu, inserted in the plasmid pSC101, can exert an influence on the efficiency of Mini-Mu DNA transposition when an induced helper Mu prophage contains a polar insertion in its semi-essential early region (SEER). The SEER of Mu is known to contain several genes that can affect DNA transposition, and our results suggest that some function(s), located in the SEER of Mu, may be required for optimizing transposition (and thus, replication) of Mu genomes from restrictive locations during the lytic cycle.

From a library of 3000 Escherichia coli clones, each containing a single, chromosomally located luxAB transcriptional gene fusion, one clone was found in which luminescence increased in the presence of 1 to 50 ppm of NiSO4. A molecular analysis revealed that the insertion occurred within the celF gene of E. coli. This gene encodes the phospho-β-glucosidase involved in cleavage of the sugars cellobiose, salicin and arbutin. Cloning and sequencing of DNA downstream of the celF gene revealed three open reading frames (potentially encoding polypeptides of 9.9, 14.1 and 28.5 kDa) that could be coexpressed with the celF gene and that may underlie the observed induction of the celF gene by nickel. A polypeptide of 26 kDa was produced when this region was placed under the control of the P tac promoter. Moreover, this region was found to be directly adjacent to, and transcribed in the opposite orientation from, the katE gene of E. coli.

The screening of environmental DNA metagenome libraries for functional activities can provide an important source of new molecules and enzymes. In this study, we identified 17 potential protease-producing clones from two metagenomic libraries derived from samples of surface sand from the Gobi and Death Valley deserts. Two of the proteases, DV1 and M30, were purified and biochemically examined. These two proteases displayed a molecular mass of 41.5 kDa and 45.7 kDa, respectively, on SDS polyacrylamide gels. Alignments with known protease sequences showed less than 55% amino acid sequence identity. These two serine proteases appear to belong to the subtilisin (S8A) family and displayed several unique biochemical properties. Protease DV1 had an optimum pH of 8 and an optimal activity at 55°C, while protease M30 had an optimum pH >11 and optimal activity at 40°C. The properties of these enzymes make them potentially useful for biotechnological applications and again demonstrate that metagenomic approaches can be useful, especially when coupled with the study of novel environments such as deserts.

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.

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luxAB gene fusions in the Escherichia coli genome were used to screen for clones displaying transcriptional changes in the presence of aluminum. One clone was found that contained a luciferase gene fusion in which transcription was increased in the presence of aluminum and which was subsequently shown to be induced by copper, iron, and nickel. Cloning of the metal-regulated gene, hybridization to the ordered phage lambda bank of the E. coli chromosome, and sequencing of DNA adjacent to the luxAB fusion revealed that the insertion occurred within the fliC (hag) gene of E. coli. This gene encodes flagellin, the filament subunit of the bacterial motility organ, and is under the control of several regulatory cascades. These results suggest that environmental metals may play a role in the regulation of the motility potential of E. coli and that this bioluminescent gene fusion clone (or derivatives thereof) may be used to prepare a biosensor for the rapid detection of metal contamination in water samples.

The c gene products from related, transposable phages Mu and D108 encode lysogenic repressors which negatively regulate transcription and transposition. Using the gel shift assay to monitor c-operator specific DNA-binding activity, the 19.5 kDa D108 c repressor was purified to homogeneity. Sequence analysis of the N-terminus confirmed the identity of the purified protein as the repressor and ascribed its ATG initiation codon to base pair 864 from the D108 left end. Analytical gel filtration and dimethyl suberimidate cross-linking of repressor at 0.1-0.5 microM concentrations revealed that the repressor protein could form oligomers in the absence of its DNA substrate. From DNase I footprinting and gel mobility shift analyses, the D108 repressor only bound to two operators (O1 and O2) which, as in Mu, flank an Integration Host Factor (IHF) binding site. In contrast to Mu, an O3 site in D108 was not found. Moreover, D108 repressor first bound operator O2, while occupancy of O1 required higher protein concentrations. The implications of these results on the D108 regulatory system are discussed.

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.

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 λ 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.

SUMMARY: Operons encoding homologous arsenic-resistance determinants (ars) have been discovered in bacterial plasmids from Gram-positive and Gram-negative, organisms, as well as in the Escherichia coli chromosome. However, evidence for this arsenic-resistance determinant in the medically and environmentally important bacterial species Pseudomonas aeruginosa is conflicting. Here the identification of a P. aeruginosa chromosomal ars operon homologue via cloning and complementation of an E. coli ars mutant is reported. The P. aeruginosa chromosomal ars operon contains three potential ORFs encoding proteins with significant sequence similarity to those encoded by the arsR, arsB and arsC genes of the plasmid-based and E. coli chromosomal ars operons. The cloned P. aeruginosa chromosomal ars operon confers augmented resistance to arsenic and antimony oxyanions in an E. coli arsB mutant and in wild-type P. aeruginosa. Expression of the operon was induced by arsenite at the mRNA level. DNA sequences homologous with this operon were detected in some, but not all, species of the genus Pseudomonas, suggesting that its conservation follows their taxonomic-based evolution.

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 excitment 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 bioavailibility.

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.Key words: arsenic resistance, Escherichia coli, transcription, gene fusions.

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.

Arsenic is a known toxic metalloid, whose trivalent and pentavalent ions can inhibit many biochemical processes. Operons which encode arsenic resistance have been found in multicopy plasmids from both gram-positive and gram-negative bacteria. The resistance mechanism is encoded from a single operon which typically consists of an arsenite ion-inducible repressor that regulates expression of an arsenate reductase and inner membrane-associated arsenite export system. Using a lacZ transcriptional gene fusion library, we have identified an Escherichia coli operon whose expression is induced by cellular exposure to sodium arsenite at concentrations as low as 5 micrograms/liter. This chromosomal operon was cloned, sequenced, and found to consist of three cistrons which we named arsR, arsB, and arsC because of their strong homology to plasmid-borne ars operons. Mutants in the chromosomal ars operon were found to be approximately 10- to 100-fold more sensitive to sodium arsenate and arsenite exposure than wild-type E. coli, while wild-type E. coli that contained the operon cloned on a ColE1-based plasmid was found to be at least 2- to 10-fold more resistant to sodium arsenate and arsenite. Moreover, Southern blotting and high-stringency hybridization of this operon with chromosomal DNAs from a number of bacterial species showed homologous sequences among members of the family Enterobacteriaceae, and hybridization was detectable even in Pseudomonas aeruginosa. These results suggest that the chromosomal ars operon may be the evolutionary precursor of the plasmid-borne operon, as a multicopy plasmid location would allow the operon to be amplified and its products to confer increased resistance to this toxic metalloid.

Ner repressors of the transposable phages Mu and D108 play a central role in regulating the expression of the early (transposase) operon and in ensuring that phage growth proceeds along a lytic pathway. The latter function is analogous to that performed by the Cro protein of phage lambda. Unlike lambda Cro, however, the structural basis of operator recognition is not known for the Ner repressors. In order to elucidate the structural features underlying operator recognition by Ner repressors, we have employed Raman spectroscopy as a probe of the solution secondary structures of both D108 Ner and Mu Ner. Additionally, we have obtained Raman spectra of the D108 Ner repressor when bound to a 61-base-pair oligodeoxynucleotide containing the 55-base-pair D108 ner binding site. Conformation-sensitive Raman bands show that both D108 and Mu Ner contain similar, highly alpha-helical (approximately 45%) secondary structures. The Raman markers also show that the substantial nonhelical secondary structure of both D108 Ner and Mu Ner is largely beta-stranded. The protein-free 61-bp D108 ner operator exhibits Raman marker bands diagnostic of an uninterrupted B DNA duplex. In the D108 Ner:DNA complex, we find the following: (i) B DNA stereochemistry is fully conserved, although with significant perturbations to the B form backbone geometry, particularly in AT-rich regions of the bound operator. (ii) The specific interactions that occur between Ner repressor and operator involve B DNA major groove sites. (iii) A small (8 +/- 3%) increase in alpha-helix content of the Ner repressor is detected upon operator binding. (iv) Finally, the local environments of many aromatic amino acids are substantially altered in the D108 Ner:DNA complex. We propose a molecular model for binding of D108 Ner to its operator that is consistent with both the present spectroscopic findings and the results of recent biochemical studies. Essential features of this model are bending of the DNA double helix and contact of operator sites with repressor domains bearing sequence homologies with the helix-turn-helix (HTH) motifs of other DNA-binding proteins. The Raman fingerprint of the Ner:DNA complex is shown to be clearly distinguishable from that of the lambda cI:DNA complex, even though both gene regulatory complexes are presumed to employ HTH recognition motifs. The unique Raman signatures observed for these repressor complexes suggest that the Raman methodology may be useful in discriminating different modes of operator recognition by the HTH motifs of regulatory proteins.

The 663 amino acid Mu transposase protein is absolutely required for Mu DNA transposition. Mutant proteins were constructed in vitro in order to locate regions of transposase that may be important for the catalysis of DNA transposition. Deletions in the A gene, which encodes the transposase, yielded two stable mutant proteins that aid in defining the end-specific DNA-binding domain. Linker insertion mutagenesis at eight sites in the Mu A gene generated two proteins, FF6 and FF14 (resulting from two and four amino acid insertions, respectively, at position 408), which were thermolabile for DNA binding in vitro at 43 degrees C. However, transposition activity in vivo was severely reduced for all mutant proteins at 37 degrees C, except those with insertions at positions 328 and 624. In addition, site-specific mutagenesis was performed to alter tyrosine 414, which is situated in a region that displays amino acid homology to the active sites of a number of nicking/closing enzymes. Tyrosine 414 may reside within an important, yet non-essential, site of transposase, as an aspartate-substituted protein had a drastically reduced frequency of transposition, while the remaining mutants yielded reduced, but substantial, frequencies of microMu transposition in vivo.

Spontaneous null mutations represent low frequency events that irreversibly and completely inactivate a gene, and can often consist of major gene alterations. To study the molecular mechanisms leading to recessive spontaneous null mutations in the human genome, we designed and tested a selection procedure in cell culture to enrich for this rare class of spontaneous mutations. The KT cell line contains the herpes simplex virus type 1 (HSV-1)_thymidine kinase (tk) gene and the neomycin-resistance gene (neo), from plasmid pSV2neoKT, integrated as a single-copy in the human tk− cell line 143B. The HSV-1 tk gene was the target for spontaneous gene inactivation, and antiviral drugs (acyclovir, trifluorothymidine and ganciclovir) were used, in combination, to provide a selective enrichment for null mutations over the background of more frequent and revertible point mutations. The tk− mutations obtained with this multiple drug selection assay appeared at a very low frequency, rarely reverted to wild-type (tk+), and the TK protein was observed only in 4.8% of these null mutants. Deletions of the entire tk gene, or its 3′ region, constituted the major class of DNA rearrangements seen in the null mutations. Additionally, one of the null mutants contained an intragenic 106-bp duplication within a 43-bp deleted region of the tk gene. We propose this mutation to be the outcome of an intragenci gene conversion event which may have been facilitated by short regions of junctional homology.

We have cloned, from the genome of HeLa cells, related 1.8-kb highly repetitive EcoRI satellite II (and III) DNA family members that displayed a high degree of TaqI and HinfI variations. Comparative sequence analysis of these cloned DNAs suggests that highly repetitive satellite DNAs may have evolved and diversified from the rapid amplification of a pentameric unit (5′ TTCCA 3′), by processes including random and non-random mutations. Base conservation, as well as mutational hotspots, were found associated with these related satellite II and III DNAs. The accumulation of C to G transversions within the pentamers, generating TaqI and HinfI sites, as well as C to T transitions at CpN dinucleotides, appear to be responsible for much of the microheterogeneity observed between related satellite II and III DNA family members.Key words: repetitive DNAs, heterochromatin, satellite DNAs, DNA sequence.

Luciferase-based biosensors are becoming increasingly used for environmental monitoring. A transcriptional fusion of the Vibrio harveyi luxAB genes (encoding bacterial luciferase) to the fliC gene of Escherichia coli was constructed and luminescence shown to be induced (in liquid media) in the presence of 1-10 micrograms/ml aluminum, but not copper, iron or nickel. Moreover, luminescence is markedly increased at pH 5.5, where aluminum is more soluble than at pH 7.0. However, aluminum also stimulated luciferase activity when the luxAB genes were located in the xyl operon. This suggests that aluminum stimulates luciferase enzyme activity in vivo. These results are specific to E. coli, as no such aluminum stimulation was observed in the luminescent bacterium V. harveyi. These results have important implications in the generalized use of these clones for environmental monitoring, where aluminum can be present at elevated concentrations.

A ColE1-based plasmid for transcriptional gene fusions was constructed that contains both the promoterless luxAB genes of Vibrio harveyi and a tet marker within the inverted repeats of a left end-truncated Tn5 element. Introduction of this plasmid into an Escherichia coli strain containing a plasmid (pTF421) that over-produces ColE1 RNA1 (and thus inhibits replication of the ColE1 plasmid) allowed selection for cells that had a single copy of the luxAB operon transposed into the chromosome beginning 5 days post-transformation. The long latent period necessary for Tn5 transposition is analogous to that found in other systems, where transposition frequencies and mutation rates increase in a time-dependent manner when selected for upon prolonged incubation on Petri dishes under bacteriostatic conditions.

Mu and D108 are related, temperate, transposable coliphages with unusual modes of DNA replication (transposition) and virion DNA maturation. These double-stranded DNA genomes replicate intrachromosomally and are matured and encapsidated linked to DNA sequences flanking the dispersed, integrated phage genomes. We have developed an in vitro system that employs crude lysates prepared from cells late in the Mu lytic cycle and that is proficient for both maturation and encapsidation of D108 DNA. Different forms of phage DNA were packaged at different efficiencies, with a circular pSC101::D108cts10 plasmid being most efficient, linearized plasmid less so, and mature virion DNA a poor substrate. The addition of purified D108 Ner protein to the reaction had no effect, whereas D108 repressor (c protein) inhibited the reaction. Escherichia coli integration host factor and D108 transposase proteins exerted an inhibitory effect on circular DNA substrates but had little effect on linear DNA packaging. This in vitro system, coupled with that developed for transposition, can now be used to biochemically dissect the protein and substrate requirements of these phages' DNA maturation pathway and the nature of the molecular switch between DNA transposition and encapsidation.

The temperate, transposable bacteriophages D108 and Mu are highly homologous, but differ in their left-end regulatory regions. We have previously cloned the gene encoding the D108 thermo-sensitive (cts) repressor under the control of thelacUV5 promoter. In this work, we report that crude protein extracts containing highly-expressed D108 repressor protect a 77 bp region of DNA, located between 863 bp and 940 bp from the D108 left-end, from both exonuclease III and DNase I hydrolysis. Nucleotide sequence analysis of this region reveals that it also contains DNA sequences homologous to the consensus DNA-binding site of theEscherichia coli protein, Integration Host Factor (IHF). Crude protein extracts containing highly-expressed IHF specifically bind to, and retard the migration of, DNA fragments containing the D108 regulatory region, and the DNA sequence which IHF protects from DNase I cleavage lies directly within the D108 repressor binding region. There are two apparent repressor-specific S1 nuclease-resistant RNAs observed during the lysogenic and lytic states. A single S1 nuclease-resistant RNA suggests that transcription from the early region promoter, Pe, may initiate at or about 1000 bp from the left-end of the D108 genome. Thus, though D108 and Mu utilize three analogous proteins (repressor, ner, and IHF) and the same apparent promoters for early gene regulation and the lytic/ lysogenic decision, the organization of these regulatory components is apparently different, suggesting different mechanisms of control of gene expression.