Ubiquitination is a highly dynamic and versatile posttranslational modification that regulates protein function, stability, and interactions. To investigate the roles of ubiquitination in a primitive eukaryotic lineage, we utilized the early-branching eukaryote Giardia intestinalis. Using a combination of biochemical, immunofluorescence-based, and proteomics approaches, we assessed the ubiquitination status during the process of differentiation in Giardia. We observed that different types of ubiquitin modifications present specific cellular and temporal distribution throughout the Giardia life cycle from trophozoites to cyst maturation. Ubiquitin signal was detected in the wall of mature cysts, and enzymes implicated in cyst wall biogenesis were identified as substrates for ubiquitination. Interestingly, inhibition of proteasome activity did not affect trophozoite replication and differentiation, while it caused a decrease in cyst viability, arguing for proteasome involvement in cyst wall maturation. Using a proteomics approach, we identified around 200 high-confidence ubiquitinated candidates that vary their ubiquitination status during differentiation. Our results indicate that ubiquitination is critical for several cellular processes in this primitive eukaryote.

Ubiquitation is an active and dynamic process in Giardia. Different types of ubiquitin modifications are present in this ancestral unicellular eukaryote and vary in terms of temporal and spatial distribution from trophozoites to cyst maturation. Using a proteomics approach, we identified around 200 high-confidence ubiquitinated candidates that vary their ubiquitination status during differentiation.

The GacS/GacA two-component regulatory system activates the production of secondary metabolites including phenazines crucial for biological control activity in Pseudomonas chlororaphis 30-84. To better understand the role of the Gac system on phenazine regulation, transcriptomic analyses were conducted by comparing the wild-type strain to a gacA mutant. RNA-seq analysis identified 771 genes under GacA control, including many novel genes. Consistent with previous findings, phenazine biosynthetic genes were significantly downregulated in a gacA mutant. The transcript abundances of phenazine regulatory genes such as phzI, phzR, iopA, iopB, rpoS, and pip also were reduced. Moreover, the transcript abundance of three noncoding RNAs (ncRNAs) including rsmX, rsmY, and rsmZ was significantly decreased by gacA mutation consistent with the presence of consensus GacA-binding sites associated with their promoters. Our results also demonstrated that constitutive expression of rsmZ from a non-gac regulated promoter resulted in complete restoration of N-acyl-homoserine lactone (AHL) and phenazine production as well as the expression of other gac-dependent secondary metabolites in gac mutants. The role of RsmA and RsmE in phenazine production also was investigated. Overexpression of rsmE, but not rsmA, resulted in decreased AHL and phenazine production in P. chlororaphis, and only a mutation in rsmE bypassed the requirement for GacA in phenazine gene expression. In contrast, constitutive expression of the phzI/phzR quorum sensing system did not rescue phenazine production in the gacA mutant, indicating the direct posttranscriptional control by Gac on the phenazine biosynthetic genes. On the basis of these results, we propose a model to illustrate the hierarchic role of phenazine regulators modulated by Gac in the control of phenazine production. The transcriptomic analysis also was used to identify additional genes regulated by GacA that may contribute to the biological control capability of strain 30-84.

Transcriptomic analysis of a gacA mutant identified 771 genes including many novel genes in Pseudomonas chlororaphis 30-84. The rsmZ and rsmE genes were linked to phenazine production. Our results also showed that constitutive expression of the quorum sensing transcriptional activator phzR, with the addition of N-acyl-homoserine lactones did not rescue phenazine production in the gacA mutant, indicating the direct posttranscriptional control by Gac of the phenazine biosynthetic genes. Figure shows proposed model of Gac/Rsm regulation of phenazines in 30-84

Bacterial community structures were evaluated in oil samples using culture-independent pyrosequencing, including oil mousses collected on sea surface and salt marshes during the Deepwater Horizon oil spill, and oil deposited in sediments adjacent to the wellhead 1 year after the spill. Phylogenetic analysis suggested that Erythrobacter, Rhodovulum, Stappia, and Thalassospira of Alphaproteobacteria were the prevailing groups in the oil mousses, which may relate to high temperatures and strong irradiance in surface Gulf waters. In the mousse collected from the leaves of Spartina alterniflora, Vibrio of Gammaproteobacteria represented 57% of the total operational taxonomic units, suggesting that this indigenous genus is particularly responsive to the oil contamination in salt marshes. The bacterial communities in oil-contaminated sediments were highly diversified. The relatively high abundance of the Methylococcus, Methylobacter, Actinobacteria, Firmicutes, and Chlorofexi bacteria resembles those found in certain cold-seep sediments with gas hydrates. Bacterial communities in the overlying water of the oil-contaminated sediment were dominated by Ralstonia of Betaproteobacteria, which can degrade small aromatics, and Saccharophagus degradans of Gammaproteobacteria, a cellulose degrader, suggesting that overlying water was affected by the oil-contaminated sediments, possibly due to the dissolution of small aromatics and biosurfactants produced during biodegradation. Overall, these results provided key information needed to evaluate oil degradation in the region and develop future bioremediation strategies.

Phylogenetic analysis suggested that Erythrobacter, Rhodovulum, Stappia and Thalassospira of Alphaproteobacteria were the prevailing groups in the sea surface oil mousse collected during the Deepwater Horizon oil spill, and this pattern may relate to high temperatures and strong irradiance in surface Gulf waters. In the salt marshes mousse, Vibrio represented 57% of the total operational taxonomic units, suggesting that this indigenous genus is particularly responsive to the oil contamination in salt marshes. In contrast, bacterial community in oil-contaminated sediments near the well head was much diversified.

We studied the effect of two commonly used fungicides on the epiphytic yeast community of strawberry. Greenhouse and field experiments were conducted applying Switch (cyprodinil plus fludioxonil) or Signum (boscalid plus pyraclostrobin) to strawberry plants. Yeasts on leaves and fruits were assessed on treated and untreated plants at several time points via plating and denaturing gradient gel electrophoresis (DGGE) analysis. The yeast counts on plates of the treated plants were similar to the control plants. Unripe fruits had 10 times larger yeast concentrations than ripe fruits or leaves. Some dominant yeast types were isolated and in vitro tests showed that they were at least 10 times less sensitive to Switch and Signum as compared with two important fungal strawberry pathogens Botrytis cinerea and Colletotrichum acutatum, which are the targets for the fungicide control. DGGE analysis showed that the applied fungicides had no effect on the composition of the yeast communities, while the growing system, strawberry tissue, and sampling time did affect the yeast communities. The yeast species most commonly identified were Cryptococcus, Rhodotorula, and Sporobolomyces. These results point toward the potential applicability of natural occurring yeast antagonists into an integrated disease control strategy for strawberry diseases.

Both conventional plating and denaturing gradient gel electrophoresis (DGGE) were used to analyse the effect of fungicides on epiphytic yeasts associated with strawberry. The results show that the main epiphytic yeasts are not affected by the fungicides and this could lead to an integrated use of epiphytic yeasts to control fungal diseases of strawberry.

A unique operon structure has been identified in the genomes of several plant- and insect-associated bacteria. The distinguishing feature of this operon is the presence of tandem hilA and hilB genes encoding dioxygenases belonging to the PF13640 and PF10014 (BsmA) Pfam families, respectively. The genes encoding HilA and HilB from Pantoea ananatis AJ13355 were cloned and expressed in Escherichia coli. The culturing of E. coli cells expressing hilA (E. coli-HilA) or both hilA and hilB (E. coli-HilAB) in the presence of l-isoleucine resulted in the conversion of l-isoleucine into two novel biogenic compounds: l-4′-isoleucine and l-4,4′-dihydroxyisoleucine, respectively. In parallel, two novel enzymatic activities were detected in the crude cell lysates of the E. coli-HilA and E. coli-HilAB strains: l-isoleucine, 2-oxoglutarate: oxygen oxidoreductase (4′-hydroxylating) (HilA) and l-4′-hydroxyisoleucine, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating) (HilB), respectively. Two hypotheses regarding the physiological significance of C-4(4′)-hydroxylation of l-isoleucine in bacteria are also discussed. According to first hypothesis, the l-isoleucine dihydroxylation cascade is involved in synthesis of dipeptide antibiotic in P. ananatis. Another unifying hypothesis is that the C-4(4′)-hydroxylation of l-isoleucine in bacteria could result in the synthesis of signal molecules belonging to two classes: 2(5H)-furanones and analogs of N-acyl homoserine lactone.

The genes encoding HilA and HilB from Pantoea ananatis AJ13355 were cloned and expressed in Escherichia coli. The culturing of E. coli cells expressing hilA (E. coli-HilA) or both hilA and hilB (E. coli-HilAB) in the presence of l-isoleucine resulted in the conversion of l-isoleucine into two novel biogenic compounds: l-4′-isoleucine and l-4,4′-dihydroxyisoleucine, respectively. In parallel, two novel enzymatic activities were detected in the crude cell lysates of the E. coli-HilA and E. coli-HilAB strains: l-isoleucine-4′-dioxygenase (HilA) and l-4′-hydroxyisoleucine-4-dioxygenase (HilB) activities, respectively.

Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that uses the overproduction of alginate, a surface polysaccharide, to form biofilms in vivo. Overproduction of alginate, also known as mucoidy, affords the bacterium protection from the host's defenses and facilitates the establishment of chronic lung infections in individuals with cystic fibrosis. Expression of the alginate biosynthetic operon is primarily controlled by the alternative sigma factor AlgU (AlgT/σ²²). In a nonmucoid strain, AlgU is sequestered by the transmembrane antisigma factor MucA to the cytoplasmic membrane. AlgU can be released from MucA via regulated intramembrane proteolysis by proteases AlgW and MucP causing the conversion to mucoidy. Pseudomonas aeruginosa strain PAO579, a derivative of the nonmucoid strain PAO1, is mucoid due to an unidentified mutation (muc-23). Using whole genome sequencing, we identified 16 nonsynonymous and 15 synonymous single nucleotide polymorphisms (SNP). We then identified three tandem single point mutations in the pilA gene (PA4525), as the cause of mucoidy in PAO579. These tandem mutations generate a premature stop codon resulting in a truncated version of PilA (PilA¹⁰⁸), with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF). Inactivation of pilA¹⁰⁸ confirmed it was required for mucoidy. Additionally, algW and algU were also required for mucoidy of PAO579. Western blot analysis indicated that MucA was less stable in PAO579 than nonmucoid PAO1 or PAO381. The mucoid phenotype and high P_algU and P_algD promoter activities of PAO579 require pilA¹⁰⁸, algW, algU, and rpoN encoding the alternative sigma factor σ⁵⁴. We also observed that RpoN regulates expression of algW and pilA in PAO579. Together, these results suggest that truncation in type IV pilin in P. aeruginosa strain PAO579 can induce mucoidy through an AlgW/AlgU-dependent pathway.

Schematic diagram of summarizing the induction of alginate production and mucoid conversion by PilA in Pseudomonas aeruginosa strain PAO579. The sigma factor RpoN is required for transcription of pilA108 and algW. PilA108 is transported to the periplasm where it activates the periplasmic protease AlgW which proteolytically degrades the anti-sigma factor MucA releasing the sequestered sigma factor AlgU.

An increasing abundance of Aphanizomenon ovalisporum in water bodies from diverse world regions has been reported in the last few years, with the majority of the isolated strains producing the toxin cylindrospermopsin (CYN), leading to a rise in ecological and health risks. The understanding of CYN synthesis is crucial in the control of CYN production. An amidinotransferase (AMDT) seems to be the first enzyme involved in the synthesis of CYN. In this study, we have cloned and overexpressed the aoaA gene from the constitutive CYN producer A. ovalisporum UAM-MAO. The recombinant purified AoaA was characterized, confirming that it is an l-arginine:glycine AMDT. It shows an optimal activity between 32 and 37°C, at pH from 8 to 9. The activity exhibits a mixed (ping-pong/sequential) kinetic mechanism, and is inhibited by the reaction product guanidine acetate (GAA) in a noncompetitive manner. Mg²⁺ stimulates AoaA activity while Co²⁺ and Mn²⁺ inhibit it. AoaA conserves the critical residues of the catalytic site and substrate specificity of AMDTs, as the previously reported AMDT from Cylindrospermopsis raciborskii Cyr. Both proteins can be included in a new group of prokaryotic AMDTs involved in CYN production.

The understanding of CYN synthesis would be crucial in control CYN production. Amidinotransferase (AMDT) seems to be the first enzyme involved in CYN synthesis. We have cloned and overexpressed the aoaA gene, which codifies for the AMDT, the constitutive CYN producer Aphanizomenon ovalisporum UAM-MAO. Purified AoaA was characterized, confirming that it is an l-arginine:glycine AMDT.

Photosynthetic bacteria can switch from planktonic lifestyle to phototrophic biofilm in mats in response to environmental changes. The mechanisms of phototrophic biofilm formation are, however, not characterized. Herein, we report a two-component system EmbRS that controls the biofilm formation in a photosynthetic member of the Burkholderiales order, the purple bacterium Rubrivivax gelatinosus. EmbRS inactivation results in cells that form conspicuous bacterial veils and fast-sinking aggregates in liquid. Biofilm analyses indicated that EmbRS represses the production of an extracellular matrix and biofilm formation. Mapping of transposon mutants that partially or completely restore the wild-type (WT) phenotype allowed the identification of two gene clusters involved in polysaccharide synthesis, one fully conserved only in Thauera sp., a floc-forming wastewater bacterium. A second two-component system BmfRS and a putative diguanylate cyclase BdcA were also identified in this screen suggesting their involvement in biofilm formation in this bacterium. The role of polysaccharides in sinking of microorganisms and organic matter, as well as the importance and the evolution of such regulatory system in phototrophic microorganisms are discussed.

Ability of the photosynthetic bacterium Rubrivivax gelatinosus to form biofilm. The EmbRS two-component system inhibits biofilm formation. Growth of the EmbRS mutant results in the formation of conspicuous bacterial veils around the toothpick scaffolds.

Despite the recent and significant increase in the study of aquatic microbial communities, little is known about the microbial diversity of complex ecosystems such as running waters. This study investigated the biodiversity of biofilm communities formed in a river with 454 Sequencing™. This river has the particularity of integrating both organic and microbiological pollution, as receiver of agricultural pollution in its upstream catchment area and urban pollution through discharges of the wastewater treatment plant of the town of Billom. Different regions of the small subunit (SSU) ribosomal RNA gene were targeted using nine pairs of primers, either universal or specific for bacteria, eukarya, or archaea. Our aim was to characterize the widest range of rDNA sequences using different sets of polymerase chain reaction (PCR) primers. A first look at reads abundance revealed that a large majority (47–48%) were rare sequences (<5 copies). Prokaryotic phyla represented the species richness, and eukaryotic phyla accounted for a small part. Among the prokaryotic phyla, Proteobacteria (beta and alpha) predominated, followed by Bacteroidetes together with a large number of nonaffiliated bacterial sequences. Bacillariophyta plastids were abundant. The remaining bacterial phyla, Verrucomicrobia and Cyanobacteria, made up the rest of the bulk biodiversity. The most abundant eukaryotic phyla were annelid worms, followed by Diatoms, and Chlorophytes. These latter phyla attest to the abundance of plastids and the importance of photosynthetic activity for the biofilm. These findings highlight the existence and plasticity of multiple trophic levels within these complex biological systems.

We performed a comprehensive analysis of bacterial and eukaryotic communities of river biofilms by pyrosequencing rDNA amplicons. The data indicated a large diversity among prokaryotes with a predominance of Proteobacteria, together with an elevated number of rare sequences. Altogether, this would indicate an important photosynthetic activity within the biofilm.

Prions are epigenetic modifiers that cause partially loss-of-function phenotypes of the proteins in Saccharomyces cerevisiae. The molecular chaperone network that supports prion propagation in the cell has seen a great progress in the last decade. However, the cellular machinery to activate or deactivate the prion states remains an enigma, largely due to insufficient knowledge of prion-regulating factors. Here, we report that overexpression of a [PSI⁺]-inducible Q/N-rich protein, Lsm4, eliminates the three major prions [PSI⁺], [URE3], and [RNQ⁺]. Subcloning analysis revealed that the Q/N-rich region of Lsm4 is responsible for the prion loss. Lsm4 formed an amyloid in vivo, which seemed to play a crucial role in the prion elimination. Fluorescence correlation spectroscopy analysis revealed that in the course of the Lsm4-driven [PSI⁺] elimination, the [PSI⁺] aggregates undergo a size increase, which ultimately results in the formation of conspicuous foci in otherwise [psi⁻]-like mother cells. We also found that the antiprion activity is a general property of [PSI⁺]-inducible factors. These data provoked a novel “unified” model that explains both prion induction and elimination by a single scheme.

Here, we report that overexpression of a [PSI⁺]-inducible Q/N-rich protein, Lsm4, eliminates the three major prions [PSI⁺], [URE3], and [RNQ⁺]. In the course of the Lsm4-driven [PSI⁺] elimination, the [PSI⁺] aggregates undergo a size increase, which ultimately results in the formation of conspicuous foci in otherwise [psi⁻]-like mother cells. We also found that the antiprion activity is a general property of [PSI⁺]-inducible factors. These data provoked a novel “unified” model that explains both prion induction and elimination by a single scheme.

The periodontal pathogen Porphyromonas gingivalis has two different lipopolysaccharides (LPSs) designated O-LPS and A-LPS, which are a conventional O-antigen polysaccharide and an anionic polysaccharide that are both linked to lipid A-cores, respectively. However, the precise mechanisms of LPS biosynthesis remain to be determined. In this study, we isolated a pigment-less mutant by transposon mutagenesis and identified that the transposon was inserted into the coding sequence PGN_2005, which encodes a hypothetical protein of P. gingivalis ATCC 33277. We found that (i) LPSs purified from the PGN_2005 mutant were shorter than those of the wild type; (ii) the PGN_2005 protein was located in the inner membrane fraction; and (iii) the PGN_2005 gene conferred Wzz activity upon an Escherichia coli wzz mutant. These results indicate that the PGN_2005 protein, which was designated WzzP, is a functional homolog of the Wzz protein in P. gingivalis. Comparison of amino acid sequences among WzzP and conventional Wzz proteins indicated that WzzP had an additional fragment at the C-terminal region. In addition, we determined that the PGN_1896 and PGN_1233 proteins and the PGN_1033 protein appear to be WbaP homolog proteins and a Wzx homolog protein involved in LPS biosynthesis, respectively.

We found a novel Porphyromonas gingivalis pigment-less mutant by transposon mutagenesis: the gene responsible was the PGN_2005 gene of strain ATCC 33277. Based on the analysis of the LPS pattern of the PGN_2005 mutant, the location and structure of the PGN_2005 protein and complementation of an Escherichia coli wzz mutant by the PGN_2005 gene, we believe that the PGN_2005 protein is Wzz of P. gingivalis, designated WzzP. In addition, in this study, we determined that the PGN_1896 and PGN_1233 proteins and the PGN_1033 protein appear to be WbaP homolog proteins and a Wzx homolog protein, respectively.

The fap operon, encoding functional amyloids in Pseudomonas (Fap), is present in most pseudomonads, but so far the expression and importance for biofilm formation has only been investigated for P. fluorescens strain UK4. In this study, we demonstrate the capacity of P. aeruginosa PAO1, P. fluorescens Pf-5, and P. putida F1 to express Fap fibrils, and investigated the effect of Fap expression on aggregation and biofilm formation. The fap operon in all three Pseudomonas species conferred the ability to express Fap fibrils as shown using a recombinant approach. This Fap overexpression consistently resulted in highly aggregative phenotypes and in increased biofilm formation. Detailed biophysical investigations of purified fibrils confirmed FapC as the main fibril monomer and supported the role of FapB as a minor, nucleating constituent as also indicated by bioinformatic analysis. Bioinformatics analysis suggested FapF and FapD as a potential β-barrel membrane pore and protease, respectively. Manipulation of the fap operon showed that FapA affects monomer composition of the final amyloid fibril, and that FapB is an amyloid protein, probably a nucleator for FapC polymerization. Our study highlights the fap operon as a molecular machine for functional amyloid formation.

Functional amyloids in Pseudomonas (Fap) are so far only described for a single environmental isolate, termed Pseudomonas fluorescens UK4. Here, we show that medical and technical important Pseudomonas strains, including P. aeruginosa PAO1, have the genetical capacity to produce Fap. Expression of Fap triggers cell autoaggregation and speed up biofilm formation.

The composition of the microbiota associated with the human ileum and colon in the early weeks of life of two preterm infants was examined, with particular emphasis on the Lactobacillus and Bifidobacterium members. Culturing work showed that bifidobacteria and lactobacilli in the ileostomy changed over time, compared with the colostomy effluent where there was far less variation. The colostomy infant was dominated by two phyla, Actinobacteria and Firmicutes, while in the ileostomy samples, Proteobacteria emerged at the expense of Actinobacteria. Bacteroidetes were only detected following the reversal of the ileostomy in the final fecal sample and were not detected in any colonic fluid samples. Clostridia levels were unstable in the colostomy fluid, suggesting that the ileostomy/colostomy itself influenced the gut microbiota, in particular the strict anaerobes. Pyrosequencing analysis of microbiota composition indicated that bifidobacteria and lactobacilli are among the dominant genera in both the ileal and colonic fluids. Bifidobacteria and lactobacilli levels were unstable in the ileostomy fluid, with large reductions in numbers and relative proportions of both observed. These decreases were characterized by an increase in proportions of Streptococcus and Enterobacteriaceae. Clostridium was detected only in the colonic effluent, with large changes in the relative proportions over time.

Pyrosequencing analysis of microbiota composition indicated that bifidobacteria and lactobacilli are among the dominant genera in both the ileal and colonic fluids. Bifidobacteria and lactobacilli levels were unstable in the ileostomy fluid, with large reductions in the numbers and relative proportions of bifidobacteria and lactobacilli observed.

The Burkholderia cepacia complex (Bcc) comprises strains with a virulence potential toward immunocompromised patients as well as plant growth–promoting rhizobacteria (PGPR). Owing to the link between quorum sensing (QS) and virulence, most studies among Bcc species have been directed toward QS of pathogenic bacteria. We have investigated the QS of B. ambifaria, a PGPR only infrequently recovered from patients. The cepI gene, responsible for the synthesis of the main signaling molecule N-octanoylhomoserine lactone (C₈-HSL), was inactivated. Phenotypes of the B. ambifaria cepI mutant we observed, such as increased production of siderophores and decreased proteolytic and antifungal activities, are in agreement with those of other Bcc cepI mutants. The cepI mutant was then used as background strain for a whole-genome transposon-insertion mutagenesis strategy, allowing the identification of 20 QS-controlled genes, corresponding to 17 loci. The main functions identified are linked to antifungal and antimicrobial properties, as we have identified QS-controlled genes implicated in the production of pyrrolnitrin, burkholdines (occidiofungin-like molecules), and enacyloxins. This study provides insights in the QS-regulated functions of a PGPR, which could lead to beneficial potential biotechnological applications.

Burkholderia ambifaria is a growth-promoting rhizobacteria that belongs to the B. cepacia complex (Bcc), also known for its virulence potential toward immunocompromised patients. To better understand the QS circuitry in this bacterium, a whole-genome transposon-insertion mutagenesis strategy has been performed. Among the QS-controlled genes identified in the study, several are implicated in antifungal and antimicrobial properties, which could lead to attractive biotechnological applications.

Identification of essential genes by construction of conditional knockouts with inducible promoters allows the identification of essential genes and creation of conditional growth (CG) mutants that are then available as genetic tools for further studies. We used large-scale transposon delivery of the rhamnose-inducible promoter, PrhaB followed by robotic screening of rhamnose-dependent growth to construct a genomic library of 106 Burkholderia cenocepacia CG mutants. Transposon insertions were found where PrhaB was in the same orientation of widely conserved, well-characterized essential genes as well as genes with no previous records of essentiality in other microorganisms. Using previously reported global gene-expression analyses, we demonstrate that PrhaB can achieve the wide dynamic range of expression levels required for essential genes when the promoter is delivered randomly and mutants with rhamnose-dependent growth are selected. We also show specific detection of the target of an antibiotic, novobiocin, by enhanced sensitivity of the corresponding CG mutant (PrhaB controlling gyrB expression) within the library. Modulation of gene expression to achieve 30–60% of wild-type growth created conditions for specific hypersensitivity demonstrating the value of the CG mutant library for chemogenomic experiments. In summary, CG mutants can be obtained on a large scale by random delivery of a tightly regulated inducible promoter into the bacterial chromosome followed by a simple screening for the CG phenotype, without previous information on gene essentiality.

We used transposon delivery of an outward-facing rhamnose-inducible promoter followed by screening of rhamnose-dependent growth to construct a genomic library of 107 Burkholderia cenocepacia conditional growth (CG) mutants. Expression from the rhamnose promoter achieved the wide dynamic range of expression levels required for essential genes. Gene regulation to obtain 30–60% of wild-type growth created conditions for specific hypersensitivity demonstrating the value of the CG mutant library for chemogenomic experiments.

We systematically studied free-living bacterial diversity within aggregations of the vestimentiferan tubeworm Ridgeia piscesae sampled from two contrasting flow regimes (High Flow and Low Flow) in the Endeavour Hydrothermal Vents Marine Protected Area (MPA) on the Juan de Fuca Ridge (Northeast Pacific). Eight samples of particulate detritus were recovered from paired tubeworm grabs from four vent sites. Most sequences (454 tag and Sanger methods) were affiliated to the Epsilonproteobacteria, and the sulfur-oxidizing genus Sulfurovum was dominant in all samples. Gammaproteobacteria were also detected, mainly in Low Flow sequence libraries, and were affiliated with known methanotrophs and decomposers. The cooccurrence of sulfur reducers from the Deltaproteobacteria and the Epsilonproteobacteria suggests internal sulfur cycling within these habitats. Other phyla detected included Bacteroidetes, Actinobacteria, Chloroflexi, Firmicutes, Planctomycetes, Verrucomicrobia, and Deinococcus–Thermus. Statistically significant relationships between sequence library composition and habitat type suggest a predictable pattern for High Flow and Low Flow environments. Most sequences significantly more represented in High Flow libraries were related to sulfur and hydrogen oxidizers, while mainly heterotrophic groups were more represented in Low Flow libraries. Differences in temperature, available energy for metabolism, and stability between High Flow and Low Flow habitats potentially explain their distinct bacterial communities.

This study is the first survey of free-living microbial diversity associated with the major foundation species at the Endeavour Hydrothermal Vents Marine Protected Area. We combined 454 tag sequencing and Sanger sequencing to characterize the composition and structure of the bacterial communities in contrasting hydrothermal habitats. While the sulphur-oxidizing genus Sulfurovum was dominant in all samples, we found significant relationships between sequence library composition and habitat type.

Insect fungiculture is practiced by ants, termites, beetles, and gall midges and it has been suggested to be widespread among plant–ants. Some of the insects engaged in fungiculture, including attine ants and bark beetles, are known to use symbiotic antibiotic-producing actinobacteria to protect themselves and their fungal cultivars against infection. In this study, we analyze the bacterial communities on the cuticles of the plant–ant genera Allomerus and Tetraponera using deep sequencing of 16S rRNA. Allomerus ants cultivate fungus as a building material to strengthen traps for prey, while Tetraponera ants cultivate fungus as a food source. We report that Allomerus and Tetraponera microbiomes contain >75% Proteobacteria and remarkably the bacterial phyla that dominate their cuticular microbiomes are very similar despite their geographic separation (South America and Africa, respectively). Notably, antibiotic-producing actinomycete bacteria represent a tiny fraction of the cuticular microbiomes of both Allomerus and Tetraponera spp. and instead they are dominated by γ-proteobacteria Erwinia and Serratia spp. Both these phyla are known to contain antibiotic-producing species which might therefore play a protective role in these ant–plant systems.

Allomerus and Tetraponera spp. ants live in symbiosis with host plants and practice cultivate fungus to use as a building material or food, respectively. The cuticles of Allomerus and Tetraponera ants are dominated by γ-proteobacteria Erwinia and Serratia spp.

Sepsis is associated with high morbidity and mortality rates worldwide. Rapid and reliable diagnostic methods are needed for efficient and evidence-based treatment of septic patients. Recently, new molecular tools have emerged to complement the conventional culture-based diagnostic methods. In this study, we used spiked whole blood samples to evaluate together two ready-to-use molecular solutions for the detection of sepsis-causing bacteria. We spiked whole blood with bacterial species relevant in sepsis and extracted bacterial DNA with the NorDiag Arrow device, using the SelectNA Blood pathogen DNA isolation kit. DNA extracts were analyzed by the polymerase chain reaction (PCR)- and microarray-based Prove-it™ Bone and Joint assay, resulting in correctly identified bacterial species with detection limits of 11–600 colony-forming unit/mL (CFU/mL). To understand the recovery losses of bacterial DNA during the sample preparation step and the capability of the PCR- and microarray-based platform to respond to the sensitivity requirements, we also determined the analytical sensitivity of the PCR and microarray platform to be 1–21 genome equivalents for the tested bacterial species. In addition, the inclusivity of the Prove-it™ Bone and Joint assay was demonstrated with methicillin-resistant Staphylococcus aureus (MRSA) clones carrying SCCmec types I, II, IV, or V and a nontypable SCCmec type. The proof-of-concept for accurate multiplex pathogen and antibacterial resistance marker detection from spiked whole blood samples was demonstrated by the selective bacterial DNA extraction method combined with the high-throughput PCR- and microarray-based platform. Further investigations are needed to study the promising potential of the concept for sensitive, semi-automated identification of sepsis-causing pathogens directly from whole blood.

The proof-of-concept for accurate multiplex pathogen and antibacterial resistance marker detection from spiked whole blood samples was demonstrated by the selective bacterial DNA extraction method combined with the high-throughput PCR and microarray-based platform.

Protein Y (PY) is an Escherichia coli cold-shock protein which has been proposed to be responsible for the repression of bulk protein synthesis during cold adaptation. Here, we present in vivo and in vitro data which clarify the role of PY and its mechanism of action. Deletion of yfiA, the gene encoding protein PY, demonstrates that this protein is dispensable for cold adaptation and is not responsible for the shutdown of bulk protein synthesis at the onset of the stress, although it is able to partially inhibit translation. In vitro assays reveal that the extent of PY inhibition changes with different mRNAs and that this inhibition is related to the capacity of PY of binding 30S subunits with a fairly strong association constant, thus stimulating the formation of 70S monomers. Furthermore, our data provide evidence that PY competes with the other ribosomal ligands for the binding to the 30S subunits. Overall these results suggest an alternative model to explain PY function during cold shock and to reconcile the inhibition caused by PY with the active translation observed for some mRNAs during cold shock.

Escherichia coli responds to cold stress by entering an acclimation phase during which protein synthesis slows down considerably with the exception of a specific set of genes (cold-shock genes) whose expression is stimulated. In this article, we have investigated in vivo and in vitro the role of PY, a protein that is associated with the ribosome throughout the cold acclimation phase. Our data indicate that protein PY can affect translation initiation but is not responsible for turning off bulk protein synthesis during the cold stress.

The complete mitochondrial genome of Rhodotorula taiwanensis RS1, an aluminum-tolerant Basidiomycota fungus, was determined and compared with the known mitochondrial genomes of 12 Basidiomycota species. The mitochondrial genome of R. taiwanensis RS1 is a circular DNA molecule of 40,392 bp and encodes the typical 15 mitochondrial proteins, 23 tRNAs, and small and large rRNAs as well as 10 intronic open reading frames. These genes are apparently transcribed in two directions and do not show syntenies in gene order with other investigated Basidiomycota species. The average G+C content (41%) of the mitochondrial genome of R. taiwanensis RS1 is the highest among the Basidiomycota species. Two introns were detected in the sequence of the atp9 gene of R. taiwanensis RS1, but not in that of other Basidiomycota species. Rhodotorula taiwanensis is the first species of the genus Rhodotorula whose full mitochondrial genome has been sequenced; and the data presented here supply valuable information for understanding the evolution of fungal mitochondrial genomes and researching the mechanism of aluminum tolerance in microorganisms.

This is, to our knowledge, the first report of the complete mitochondrial genome sequence of the genus Rhodotorula, and contains comparison with known 12 mitochondrial genomes of Basidiomycota species. The mitochondrial genome of Rhodotorula taiwanensis RS1 has two unique features, that is, relatively higher G+C content and two intron insertions into the atp9 gene. This study will supply basic information for understanding the evolution of fungal mitochondrial genomes and researching the mechanism of aluminum tolerance in microorganisms.

The formation of a Pseudomonas aeruginosa biofilm, a complex structure enclosing bacterial cells in an extracellular polymeric matrix, is responsible for persistent infections in cystic fibrosis patients leading to a high rate of morbidity and mortality. The protective environment created by the tridimensional structure reduces the susceptibility of the bacteria to conventional antibiotherapy. Cationic steroid antibiotics (CSA)-13, a nonpeptide mimic of antimicrobial peptides with antibacterial activity on planktonic cultures, was evaluated for its ability to interact with sessile cells. Using confocal laser scanning microscopy, we demonstrated that the drug damaged bacteria within an established biofilm showing that penetration did not limit the activity of this antimicrobial agent against a biofilm. When biofilms were grown during exposure to shear forces and to a continuous medium flow allowing the development of robust structures with a complex architecture, CSA-13 reached the bacteria entrapped in the biofilm within 30 min. The permeabilizing effect of CSA-13 could be associated with the death of the bacteria. In static conditions, the compound did not perturb the architecture of the biofilm. This study confirms the potential of CSA-13 as a new strategy to combat persistent infections involving biofilms formed by P. aeruginosa.

Persistent Pseudomonas aeruginosa respiratory infections are linked to the formation of a biofilm and are a common cause of morbidity and mortality in cystic fibrosis patients. The worldwide increase resistance to conventional antibiotics has motived the search for new strategies to combat chronic infections. Confocal laser scanning microscopy was used in this study to demonstrate the ability of the drug to kill bacteria within a complex and robust biofilm. Cationic steroid antibiotics (CSA)-13 offers promising potential to treat not only acute infections caused by planktonic bacteria but also chronic infections involving biofilms.

Bacteroides fragilis is the most frequent opportunistic pathogen isolated from anaerobic infections. However, there is a paucity of information regarding the genetic and molecular aspects of gene expression of its virulence factors during extra-intestinal infections. A potential virulence factor that has received little attention is the ability of B. fragilis to produce hemolysins. In this study, an implanted perforated table tennis “ping-pong” ball was used as an intra-abdominal artificial abscess model in the rat. This procedure provided sufficient infected exudate for gene expression studies in vivo. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify the relative expression of hlyA, hlyB, hlyC, hlyD, hlyE, hlyF, hlyG, and hlyIII mRNAs. The hlyA mRNA was induced approximately sixfold after 4 days postinfection compared with the mRNA levels in the inoculum culture prior to infection. The hlyB mRNA increased approximately sixfold after 4 days and 12-fold after 8 days postinfection. Expression of hlyC mRNA increased sixfold after 1 day, 45-fold after 4 days, and 16-fold after 8 days postinfection, respectively. The hlyD and hlyE mRNAs were induced approximately 40-fold and 30-fold, respectively, after 4-days postinfection. The hlyF expression increased approximately threefold after 4-days postinfection. hlyG was induced approximately fivefold after 4 and 8 days postinfection. The hlyIII mRNA levels had a steady increase of approximately four-, eight-, and 12-fold following 1, 4, and 8 days postinfection, respectively. These findings suggest that B. fragilis hemolysins are induced and differentially regulated in vivo. Both parent and hlyBA mutant strains reached levels of approximately 3–8 × 10⁹ cfu/mL after 1 day postinfection. However, the hlyBA mutant strain lost 2 logs in viable cell counts compared with the parent strain after 8 days postinfection. This is the first study showing HlyBA is a virulence factor which plays a role in B. fragilis survival in an intra-abdominal abscess model.

Bacteroides fragilis hemolysins are expressed and regulated in vivo. The dual-hemolysin component HlyBA is required for survival in an intra-abdominal infection model. This is the first study showing that hemolysins are virulence factor in B. fragilis, the most frequent anaerobe isolated from human infection.

The thallus surface of the brown macroalga Fucus vesiculosus is covered by a specific biofilm community. This biofilm supposedly plays an important role in the interaction between host and environment. So far, we know little about compositional or functional shifts of this epibiotic bacterial community under changing environmental conditions. In this study, the response of the microbiota to different temperatures with respect to cell density and community composition was analyzed by nonculture-based methods (denaturing gradient gel electrophoresis and 454 pyrosequencing of the 16S rRNA gene). Redundancy analysis showed that despite high variability among host individuals temperature accounted for 20% of the variation in the bacterial community composition, whereas cell density did not differ between groups. Across all samples, 4341 bacterial operational taxonomic units (OTUs) at a 97% similarity level were identified. Eight percent of OTUs were significantly correlated with low, medium, and high temperatures. Notably, the family Rhodobacteraceae increased in relative abundance from 20% to 50% with increasing temperature. OTU diversity (evenness and richness) was higher at 15°C than at the lower and higher temperatures. Considering their known and presumed ecological functions for the host, change in the epibacterial community may entail shifts in the performance of the host alga.

This study explores the compositional shifts and the diversity of the bacterial community associated with the brown macroalga Fucus vesiculosus under different temperature conditions using fingerprinting and pyrosequencing methods.

Integral membrane proteins with α-helical transmembrane segments (TMS) are known to play important and diverse roles in prokaryotic cell physiology. The net hydrophobicity of TMS directly corresponds to the observed difficulties in expressing and purifying these proteins, let alone producing sufficient yields for structural studies using two-/three-dimensional (2D/3D) crystallographic or nuclear magnetic resonance methods. To gain insight into the function of these integral membrane proteins, topological mapping has become an important tool to identify exposed and membrane-embedded protein domains. This approach has led to the discovery of protein tracts of functional importance and to the proposition of novel mechanistic hypotheses. In this review, we synthesize the various methods available for topological mapping of α-helical integral membrane proteins to provide investigators with a comprehensive reference for choosing techniques suited to their particular topological queries and available resources.

In this review, we synthesize the various methods available for topological mapping of α-helical integral membrane proteins to provide investigators with a comprehensive reference for choosing techniques suited to their particular topological queries and available resources.