Benoit Cournoyer

A tale of two oxidation states: bacterial colonization of arsenic-rich environments.

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments—including ground and surface waters—from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the β-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth.

Burkholderia graminis sp. nov., a rhizospheric Burkholderia species, and reassessment of [Pseudomonas] phenazinium, [Pseudomonas] pyrrocinia and [Pseudomonas] glathei as Burkholderia

In a survey of soil and wheat or maize rhizoplane bacteria isolated using a medium containing azelaic acid and tryptamine as sole carbon and nitrogen sources, respectively, a large proportion of Burkholderia-like bacteria were found. Among them, a homogeneous group of strains was identifiable based on phenotypic properties, fatty acid composition, DNA-DNA hybridizations and 16S rDNA sequences. According to molecular data, this group belongs to the genus Burkholderia but its weak similarity to previously described species suggests that it belongs to a novel species. Closest 16S rDNA phylogenetic neighbours of this species are Burkholderia caryophylli and two previously named Pseudomonas species which clearly appear to be part of the Burkholderia genus and were thus named Burkholderia glathei comb. nov. and Burkholderia phenazinium comb. nov. Strains of the new species are oxidase- and catalase-positive, produce indole and gelatinase, and use L-xylose, lactose, rhamnose, trehalose, D-lyxose, L-arabitol, xylitol and D-raffinose as sole carbon source. This novel taxon is named Burkholderia graminis. In the course of this study, [Pseudomonas] pyrrocinia also proved to be a member of the Burkholderia genus.

Antiadhesive properties of glycoclusters against Pseudomonas aeruginosa lung infection

Pseudomonas aeruginosa lung infections are a major cause of death in cystic fibrosis and hospitalized patients. Treating these infections is becoming difficult due to the emergence of conventional antimicrobial multiresistance. While monosaccharides have proved beneficial against such bacterial lung infection, the design of several multivalent glycosylated macromolecules has been shown to be also beneficial on biofilm dispersion. In this study, calix[4]arene-based glycoclusters functionalized with galactosides or fucosides have been synthesized. The characterization of their inhibitory properties on Pseudomonas aeruginosa aggregation, biofilm formation, adhesion on epithelial cells, and destruction of alveolar tissues were performed. The antiadhesive properties of the designed glycoclusters were demonstrated through several in vitro bioassays. An in vivo mouse model of lung infection provided an almost complete protection against Pseudomonas aeruginosa with the designed glycoclusters.

Methylation of Inorganic and Organic Selenium by the Bacterial Thiopurine Methyltransferase

Escherichia coli cells expressing the tpm gene encoding the bacterial thiopurine methyltransferase (bTPMT) are shown to methylate selenite and (methyl)selenocysteine into dimethylselenide (DMSe) and dimethyldiselenide (DMDSe). E. coli cells expressing tpm from a gene library cosmid clone (harboring a Pseudomonas syringae insert of about 20 kb) also methylated selenate into DMSe and DMDSe. bTPMT is the first methyltransferase shown to be involved in the methylation of these selenium derivatives.

Characterization of the 20S proteasome from the actinomycete Frankia

Frankia is an actinomycete that fixes atmospheric nitrogen in symbiotic association with the root systems of a variety of non‐leguminous plants, denominated actinorhizal plants. Information on the biology of proteolysis in Frankia is almost non‐existent as it is extremely difficult to grow this organism. We have purified 20S proteasomes from Frankia strain ACN14a/ts‐r. It is composed of one α‐subunit and one β‐subunit, which assemble into the canonical structure of four rings of seven subunits each. The enzyme displayed a chymotrypsin‐like activity against synthetic substrates and was sensitive to lactacystin, a specific proteasome inhibitor. Analysis of the structural genes and the flanking regions revealed a similar organization to Rhodococcus erythropolis, Mycobacterium tuberculosis and Streptomyces coelicolor and showed that the β‐subunit is encoded with a 52‐amino‐acid propeptide that is cleaved off in the course of the assembly. We report also for the first time the in vitro assembly of chimeric proteasomes composed of Frankia and Rhodococcus erythropolis subunits, which are correctly assembled and proteolytically active.

Antibiotic and metal resistance among hospital and outdoor strains of Pseudomonas aeruginosa

Phenotypic analyses of antibiotic and metal resistance of a collection of 130 strains of Pseudomonas aeruginosa from various outdoor (i.e. soil, water, animals) and hospital (environment, patients, individuals with cystic fibrosis) settings were performed. Resistance was scored according to the origin of the strains and their likely exposure to antibiotics and chemicals. Most of the 76 outdoor strains showed a wild-type antibiotic resistance phenotype, i.e. resistance to minocycline and trimethoprim-sulfamethoxazole. Sixty percent of hospital strains showed a multiresistance phenotype (from 3 to 16 antibiotics) and confirmed that frequent exposure to antibiotics favored selection and maintenance of antibiotic resistance in P. aeruginosa. Twelve percent of outdoor strains naturally exposed to antiseptics and hydrocarbons showed significant resistance profiles, suggesting that chemical contaminants could contribute to selection of antibiotic resistance. For metal resistance, outdoor strains were more frequently resistant to zinc and cadmium, whereas hospital strains were more frequently resistant to mercury and copper. Differences in metal resistance between the 130 strains investigated were not related to previously characterized processes such as those implicating czcA, involved in cadmium, zinc, and cobalt resistance, or copA and copB, involved in copper resistance. Regulatory or new processes were likely to have contributed to the observed variations. Strains showing strong resistance to antibiotics were the least resistant to metals, and inversely. The lack of significant correlations between antibiotic and metal resistance suggests involvement of distinct processes that are rarely co-selected. The effects of the P. aeruginosa collection size and multi-factorial selective pressure on data sets are discussed.

Freshwater bacteria can methylate selenium through the thiopurine methyltransferase pathway.

ABSTRACT Involvement of the bacterial thiopurine methyltransferase (bTPMT) in natural selenium methylation by freshwater was investigated. A freshwater environment that had no known selenium contamination but exhibited reproducible emission of dimethyl selenide (DMSe) or dimethyl diselenide (DMDSe) when it was supplemented with an organic form of selenium [(methyl)selenocysteine] or an inorganic form of selenium (sodium selenite) was used. The distribution of the bTPMT gene (tpm) in the microflora was studied. Freshwater bacteria growing on 10 μM sodium selenite and 10 μM sodium selenate were isolated, and 4.5 and 10% of the strains, respectively, were shown by colony blot hybridization to hybridize with a Pseudomonas syringae tpm DNA probe. Ribotyping showed that these strains are closely related. The complete rrs sequence of one of the strains, designated Hsa.28, was obtained and analyzed. Its closest phyletic neighbor was found to be the Pseudomonas anguilliseptica rrs sequence. The Hsa.28 strain grown with sodium selenite or (methyl)selenocysteine produced significant amounts of DMSe and DMDSe. The Hsa.28 tpm gene was isolated by genomic DNA library screening and sequencing. BLASTP comparisons of the deduced Hsa.28 bTPMT sequence with P. syringae, Pseudomonas aeruginosa, Vibrio cholerae, rat, and human thiopurine methyltransferase sequences revealed that the levels of similarity were 52 to 71%. PCR-generated Escherichia coli subclones containing the Hsa.28 tpm open reading frame were constructed. E. coli cells harboring the constructs and grown with sodium selenite or (methyl)selenocysteine produced significant levels of DMSe and DMDSe, confirming that the gene plays a role in selenium methylation. The effect of strain Hsa.28 population levels on freshwater DMSe and DMDSe emission was investigated. An increase in the size of the Hsa.28 population was found to enhance significantly the emission of methyl selenides by freshwater samples supplemented with sodium selenite or (methyl)selenocysteine. These data suggest that bTPMT can play a role in natural freshwater selenium methylation processes.

Infrequent Finding of Metallo-β-Lactamase VIM-2 in Carbapenem-Resistant Pseudomonas aeruginosa Strains from Croatia

ABSTRACT One hundred sixty-nine nonreplicate imipenem-resistant Pseudomonas aeruginosa strains isolated in a large hospital on the coastal region of Croatia were studied. The most active antibiotics were colistin and amikacin. Most of the isolates were multiresistant. The most prevalent serotype was O12, followed by O11. Six strains carried the blaVIM-2 gene located in a novel class 1 integron composed in its variable part of the blaVIM-2-blaoxa-10-ΔqacF-aacA4 genes. Metallo-β-lactamase-producing strains belonged to sequence types ST235 and ST111.

Characterization of a spontaneous thiostrepton-resistant Frankia alni infective isolate using PCR-RFLP of nif and glnII genes

Abstract A spontaneous thiostrepton-resistant isolate of Frankia alni strain ACN14a is described. This isolate, named ACN14tsr, is infective and effective when in association with Alnus glutinosa . The resistance to thiostrepton is stable and maintained after growth in the absence of the antibiotic. The isolate was shown to be related to its parent strain by a three step PCR (polymerase chain reaction)-based method using nif and gln II genes. Frankia -specific PCR amplification of the gln II gene was used as a taxonomic marker of the genus. The defined PCR primers allowed amplification of the gln II gene from all Frankia strains tested so far but did not detect gln II in Geodermatophilus obscurus, Blastococcus aggregatus and the Actinoplanetes. This approach discriminated the thiostrepton-resistant Frankia isolate from closely related genera of the Actinomycetes. RFLP (restriction fragment length polymorphism) analysis of PCR fragments from the nif HD operon of various Frankia species then reliably related the isolate to Frankia alni . Finally, RFLP analysis of PCR fragments from the gln II gene allowed identification of the parent strain of the thiostrepton-resistant isolate. This is the first description of a subspecific characterization of strains of Frankia alni using amplified genes and RFLP patterns.

Identification of Stenotrophomonas maltophilia strains isolated from environmental and clinical samples: a rapid and efficient procedure

Aims:  Aim of the study is to identify accurately Stenotrophomonas maltophilia isolates recovered from environmental and clinical samples.