Genome sequence of PSonyx, a singleton bacteriophage infecting Corynebacterium glutamicum.

PSonyx is a newly isolated phage that infects Corynebacterium glutamicum. This siphovirus was isolated from a French pond in the south of Paris by students from Paris-Saclay University. Its 80,277-bp singleton genome carries 136 protein-coding genes and 5 tRNAs.

5,12-Dihydroindolo[3,2-a]Carbazole Derivatives-Based Water Soluble Photoinitiators for 3D Antibacterial Hydrogels Preparation.

Indolo[3,2-a]carbazole alkaloids have drawn a growing interest in recent years owing to their potential electrical and optical properties. With 5,12-dihydroindolo[3,2-a]carbazole serving as the scaffold, two novel carbazole derivatives are synthesized in this study. Both compounds are extremely soluble in water, with solubility surpassing 7% in weight. Intriguingly, the introduction of aromatic substituents contributed to drastically reduce the π-stacking ability of carbazole derivatives, while the presence of the sulfonic acid groups enables the resulting carbazoles remarkably soluble in water, allowing them to be used as especially efficient water-soluble PIs in conjunction with co-initiators, i.e., triethanolamine and the iodonium salt, respectively, employed as electron donor and acceptor. Surprisingly, multi-component photoinitiating systems based on these synthesized carbazole derivatives could be used for the in situ preparation of hydrogels containing silver nanoparticles via laser write procedure with a light emitting diode (LED)@405 nm as light source, and the produced hydrogels display antibacterial activity against Escherichia coli.

Novel liposome-like assemblies composed of phospholipid-porphyrin conjugates with photothermal and photodynamic activities against bacterial biofilms.

Phospholipid-Porphyrin (PL-Por) conjugates are unique building blocks that can self assemble into liposome-like structures with improved photophysical properties compared to their monomeric counterparts. The high packing density of porphyrin moieties enables these assemblies to exhibit high photothermal conversion efficiency as well as photodynamic activity. Thus, PL-Por conjugates assemblies can be used for photodynamic therapy (PDT) and photothermal therapy (PTT) applications against resistant bacteria and biofilms. In order to tune the PD/PT properties of such nanosystems, we developed six different supramolecular assemblies composed of newly synthesized PL-Por conjugates bearing either pheophorbide-a (PhxLPC) or pyropheophorbide-a (PyrxLPC) photosensitizers (PSs) for combined PDT/PTT against planktonic bacteria and their biofilms. In this study, the influence of the chemical structure of the phospholipid backbone as well as that of the PS on the photothermal conversion efficiency, the photodynamic activity and the stability of these assemblies in biological medium were determined. Then their antimicrobial efficiency was assessed on S. aureus and P. aeruginosa planktonic cultures and biofilms. The two studied systems show almost the same photothermal effect against planktonic cultures and biofilms of S. aureus and P. aeruginosa. However, PhxLPC vesicles exhibit superior photodynamic activity, making them the best combination for PTT/PDT. Such results highlight the higher potential of the photodynamic activity of PL-Por nanoassemblies compared to their photothermal conversion in combating bacterial infections.

Unveiling Ecological and Genetic Novelty within Lytic and Lysogenic Viral Communities of Hot Spring Phototrophic Microbial Mats.

Viruses exert diverse ecosystem impacts by controlling their host community through lytic predator-prey dynamics. However, the mechanisms by which lysogenic viruses influence their host-microbial community are less clear. In hot springs, lysogeny is considered an active lifestyle, yet it has not been systematically studied in all habitats, with phototrophic microbial mats (PMMs) being particularly not studied. We carried out viral metagenomics following in situ mitomycin C induction experiments in PMMs from Porcelana hot spring (Northern Patagonia, Chile). The compositional changes of viral communities at two different sites were analyzed at the genomic and gene levels. Furthermore, the presence of integrated prophage sequences in environmental metagenome-assembled genomes from published Porcelana PMM metagenomes was analyzed. Our results suggest that virus-specific replicative cycles (lytic and lysogenic) were associated with specific host taxa with different metabolic capacities. One of the most abundant lytic viral groups corresponded to cyanophages, which would infect the cyanobacteria Fischerella, the most active and dominant primary producer in thermophilic PMMs. Likewise, lysogenic viruses were related exclusively to chemoheterotrophic bacteria from the phyla Proteobacteria, Firmicutes, and Actinobacteria. These temperate viruses possess accessory genes to sense or control stress-related processes in their hosts, such as sporulation and biofilm formation. Taken together, these observations suggest a nexus between the ecological role of the host (metabolism) and the type of viral lifestyle in thermophilic PMMs. This has direct implications in viral ecology, where the lysogenic-lytic switch is determined by nutrient abundance and microbial density but also by the metabolism type that prevails in the host community. IMPORTANCE Hot springs harbor microbial communities dominated by a limited variety of microorganisms and, as such, have become a model for studying community ecology and understanding how biotic and abiotic interactions shape their structure. Viruses in hot springs are shown to be ubiquitous, numerous, and active components of these communities. However, lytic and lysogenic viral communities of thermophilic phototrophic microbial mats (PMMs) remain largely unexplored. In this work, we use the power of viral metagenomics to reveal changes in the viral community following a mitomycin C induction experiment in PMMs. The importance of our research is that it will improve our understanding of viral lifestyles in PMMs via exploring the differences in the composition of natural and induced viral communities at the genome and gene levels. This novel information will contribute to deciphering which biotic and abiotic factors may control the transitions between lytic and lysogenic cycles in these extreme environments.

Biocorrosion on Nanofilms Induces Rapid Bacterial Motions via Iron Dissolution.

Stability and reactivity of solid metal or mineral surfaces in contact with bacteria are critical properties for development of biocorrosion protection and for understanding bacteria-solid environmental interactions. Here, we opted to work with nanosheets of iron nanolayers offering arbitrarily large and stable areas of contact that can be simply monitored by optical means. We focused our study on the sediments' bacteria, the strain Shewanella oneidensis WT MR-1, that served as models for previous research on electroactivity and iron-reduction effects. Data show that a sudden uniform corrosion appeared after an early electroactive period without specific affinities and that iron dissolution induced rapid bacterial motions. By extending the approach to mutant strains and three bacterial species, we established a correlation between corrosion onset and oxygen-depletion combined with iron reduction and demonstrated bacteria's extraordinary ability to transform their solid environments.

Variation of bacterial biodiversity from saline soils and estuary sediments present near the Mediterranean Sea coast of Camargue (France).

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 (Rhône 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 1 g 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.

Advanced microRNA-based cancer diagnostics using amplified time-gated FRET.

MicroRNAs (miRNAs) play an important role in cellular functions and in the development and progression of cancer. Precise quantification of endogenous miRNAs from different clinical patient and control samples combined with a one-to-one comparison to standard technologies is a challenging but necessary endeavor that is largely neglected by many emerging fluorescence technologies. Here, we present a simple, precise, sensitive, and specific ratiometric assay for absolute quantification of miRNAs. Isothermally amplified time-gated Förster resonance energy transfer (TG-FRET) between Tb donors and dye acceptors resulted in miRNA assays with single-nucleotide variant specificity and detection limits down to 4.2 ± 0.5 attomoles. Quantification of miR-21 from human tissues and plasma samples revealed the relevance for breast and ovarian cancer diagnostics. Analysis of miR-132 and miR-146a from acute monocytic leukemia cells (THP-1) demonstrated the broad applicability to different miRNAs and other types of clinical samples. Direct comparison to the gold standard RT-qPCR showed advantages of amplified TG-FRET concerning precision and specificity when quantifying low concentrations of miRNAs as required for diagnostic applications. Our results demonstrate that a careful implementation of rolling circle amplification and TG-FRET into one straightforward nucleic acid detection method can significantly advance the possibilities of miRNA-based cancer diagnostics and research.

Mechanical Behavior of a Bacillus subtilis Pellicle.

Bacterial biofilms consist of a complex network of biopolymers embedded with microorganisms, and together these components form a physically robust structure that enables bacteria to grow in a protected environment. This structure can help unwanted biofilms persist in situations ranging from chronic infection to the biofouling of industrial equipment, but under certain circumstances it can allow the biofilm to disperse and colonize new niches. Mechanical properties are therefore a key aspect of biofilm life. In light of the recently discovered growth-induced compressive stress present within a biofilm, we studied the mechanical behavior of Bacillus subtilis pellicles, or biofilms at the air-liquid interface, and tracked simultaneously the force response and macroscopic structural changes during elongational deformations. We observed that pellicles behaved viscoelastically in response to small deformations, such that the growth-induced compressive stress was still present, and viscoplastically at large deformations, when the pellicles were under tension. In addition, by using particle imaging velocimetry we found that the pellicle deformations were nonaffine, indicating heterogeneous mechanical properties with the pellicle being more pliable near attachment surfaces. Overall, our results indicate that we must consider not only the viscoelastic but also the viscoplastic and mechanically heterogeneous nature of these structures to understand biofilm dispersal and removal.

The bacteria and bacteriophages from a Mesquite Flats site of the Death Valley desert.

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.

Elasticity and wrinkled morphology of Bacillus subtilis pellicles.

Wrinkled morphology is a distinctive phenotype observed in mature biofilms produced by a great number of bacteria. Here we study the formation of macroscopic structures (wrinkles and folds) observed during the maturation of Bacillus subtilis pellicles in relation to their mechanical response. We show how the mechanical buckling instability can explain their formation. By performing simple tests, we highlight the role of confining geometry and growth in determining the symmetry of wrinkles. We also experimentally demonstrate that the pellicles are soft elastic materials for small deformations induced by a tensile device. The wrinkled structures are then described by using the equations of elastic plates, which include the growth process as a simple parameter representing biomass production. This growth controls buckling instability, which triggers the formation of wrinkles. We also describe how the structure of ripples is modified when capillary effects are dominant. Finally, the experiments performed on a mutant strain indicate that the presence of an extracellular matrix is required to maintain a connective and elastic pellicle.

Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts.

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.

Reductive dechlorination of tetrachloroethene by a stepwise catalysis of different organohalide respiring bacteria and reductive dehalogenases.

The enrichment culture SL2 dechlorinating tetrachloroethene (PCE) to ethene with strong trichloroethene (TCE) accumulation prior to cis-1,2-dichloroethene (cis-DCE) formation was analyzed for the presence of organohalide respiring bacteria and reductive dehalogenase genes (rdhA). Sulfurospirillum-affiliated bacteria were identified to be involved in PCE dechlorination to cis-DCE whereas "Dehalococcoides"-affiliated bacteria mainly dechlorinated cis-DCE to ethene. Two rdhA genes highly similar to tetrachloroethene reductive dehalogenase genes (pceA) of S. multivorans and S. halorespirans were present as well as an rdhA gene very similar to the trichloroethene reductive dehalogenase gene (tceA) of "Dehalococcoides ethenogenes" strain 195. A single strand conformation polymorphism (SSCP) method was developed allowing the simultaneous detection of the three rdhA genes and the estimation of their abundance. SSCP analysis of different SL2 cultures showed that one pceA gene was expressed during PCE dechlorination whereas the second was expressed during TCE dechlorination. The tceA gene was involved in cis-DCE dechlorination to ethene. Analysis of the internal transcribed spacer region between the 16S and 23S rRNA genes revealed two distinct sequences originating from Sulfurospirillum suggesting that two Sulfurospirillum populations were present in SL2. Whether each Sulfurospirillum population was catalyzing a different dechlorination step could however not be elucidated.

The use of genomic signature distance between bacteriophages and their hosts displays evolutionary relationships and phage growth cycle determination.

BACKGROUND: Bacteriophage classification is mainly based on morphological traits and genome characteristics combined with host information and in some cases on phage growth lifestyle. A lack of molecular tools can impede more precise studies on phylogenetic relationships or even a taxonomic classification. The use of methods to analyze genome sequences without the requirement for homology has allowed advances in classification. RESULTS: Here, we proposed to use genome sequence signature to characterize bacteriophages and to compare them to their host genome signature in order to obtain host-phage relationships and information on their lifestyle. We analyze the host-phage relationships in the four most representative groups of Caudoviridae, the dsDNA group of phages. We demonstrate that the use of phage genomic signature and its comparison with that of the host allows a grouping of phages and is also able to predict the host-phage relationships (lytic vs. temperate). CONCLUSIONS: We can thus condense, in relatively simple figures, this phage information dispersed over many publications.

Indications for acquisition of reductive dehalogenase genes through horizontal gene transfer by Dehalococcoides ethenogenes strain 195.

The genome of Dehalococcoides ethenogenes strain 195, an anaerobic dehalorespiring bacterium, contains 18 copies of putative reductive dehalogenase genes, including the well-characterized tceA gene, whose gene product functions as the key enzyme in the environmentally important dehalorespiration process. The genome of D. ethenogenes was analyzed using a bioinformatic tool based on the frequency of oligonucleotides. The results in the form of a genomic signature revealed several local disruptions of the host signature along the genome sequence. These fractures represent DNA segments of potentially foreign origin, so-called atypical regions, which may have been acquired by an ancestor through horizontal gene transfer. Most interestingly, 15 of the 18 reductive dehalogenase genes, including the tceA gene, were found to be located in these regions, strongly indicating the foreign nature of the dehalorespiration activity. The GC content and the presence of recombinase genes within some of these regions corroborate this hypothesis. A hierarchical classification of the atypical regions containing the reductive dehalogenase genes indicated that these regions were probably acquired by several gene transfer events.

Isolation and characterization of Tn-Dha1, a transposon containing the tetrachloroethene reductive dehalogenase of Desulfitobacterium hafniense strain TCE1.

A new 9.9 kb catabolic transposon, Tn-Dha1, containing the gene responsible for tetrachloroethene (PCE) reductive dechlorination activity, was isolated from Desulfitobacterium hafniense strain TCE1. Two fully identical copies of the insertion sequence ISDha1, a new member of the IS256 family, surround the gene cluster pceABCT, a truncated gene for another transposase and a short open reading frame with homology to a member of the twin-arginine transport system (tatA). Evidence was obtained by Southern blot for an alternative form of the transposon element as a circular molecule containing only one copy of ISDha1. This latter structure most probably represents a dead-end product of the transposition of Tn-Dha1. Strong indications for the transposition activity of ISDha1 were given by polymerase chain reaction (PCR) amplification and sequencing of the intervening sequence located between both inverted repeats (IR) of ISDha1 (IR junction). A stable genomic ISDha1 tandem was excluded by quantitative real-time PCR. Promoter mapping of the pceA gene, encoding the reductive dehalogenase, revealed the presence of a strong promoter partially encoded in the right inverted repeat of ISDha1. A sequence comparison with pce gene clusters from Desulfitobacterium sp. strains PCE-S and Y51 and from Dehalobacter restrictus, all of which show 100% identity for the pceAB genes, indicated that both Desulfitobacterium strains seem to possess the same transposon structure, whereas only the pceABCT gene cluster is conserved in D. restrictus.

Development of degenerate and specific PCR primers for the detection and isolation of known and putative chloroethene reductive dehalogenase genes.

Degenerate and specific PCR primers were designed for the detection of chloroethene reductive dehalogenases (CE-RDase), the key enzymes of chloroethene dehalorespiration, based on sequence information of three CE-RDases and three chlorophenol (CP) RDases. For the design of the degenerate primers, seven conserved amino-acid blocks identified with different bioinformatic tools were used. For one block degenerate, primers containing a 5'-consensus clamp region specific for CE-RDases and a 3'-end degenerate core region specific for RDases in general were designed using the Consensus-Degenerate Hybrid Oligonucleotide Primer (CDHOP) design method. Applying the degenerate primers to genomic DNA of Sulfurospirillum multivorans strain K, Dehalobacter restrictus strain PER-K23, and Desulfitobacterium sp. strain PCE1 led to the isolation of the known CE-RDase genes and three new genes encoding putative reductive dehalogenases that cluster with CE-RDases and not with CP-RDases. In addition, primers designed to be specific for the three known CE-RDase genes, namely pceA of S. multivorans, pceA of D. restrictus, and tceA of Dehalococcoides ethenogenes were successfully tested on genomic DNA of different chloroethene-dehalorespiring bacteria. Nested PCR using degenerate primers followed by a PCR with specific primers allowed a sensitive detection of only 10(2) copies per reaction.

Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus.

The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 +/- 1 kDa, whereas the native molecular mass was 71 +/- 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-beta-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 +/- 0.1 mol of cobalamin, 0.6 +/- 0.02 mol of cobalt, 7.1 +/- 0.6 mol of iron, and 5.8 +/- 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 +/- 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The K(m) values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 +/- 3.2, 23.7 +/- 5.2, and 47 +/- 10 micro M, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.