Rob Van Houdt

The Complete Genome Sequence of Cupriavidus metallidurans Strain CH34, a Master Survivalist in Harsh and Anthropogenic Environments

Many bacteria in the environment have adapted to the presence of toxic heavy metals. Over the last 30 years, this heavy metal tolerance was the subject of extensive research. The bacterium Cupriavidus metallidurans strain CH34, originally isolated by us in 1976 from a metal processing factory, is considered a major model organism in this field because it withstands milli-molar range concentrations of over 20 different heavy metal ions. This tolerance is mostly achieved by rapid ion efflux but also by metal-complexation and -reduction. We present here the full genome sequence of strain CH34 and the manual annotation of all its genes. The genome of C. metallidurans CH34 is composed of two large circular chromosomes CHR1 and CHR2 of, respectively, 3,928,089 bp and 2,580,084 bp, and two megaplasmids pMOL28 and pMOL30 of, respectively, 171,459 bp and 233,720 bp in size. At least 25 loci for heavy-metal resistance (HMR) are distributed over the four replicons. Approximately 67% of the 6,717 coding sequences (CDSs) present in the CH34 genome could be assigned a putative function, and 9.1% (611 genes) appear to be unique to this strain. One out of five proteins is associated with either transport or transcription while the relay of environmental stimuli is governed by more than 600 signal transduction systems. The CH34 genome is most similar to the genomes of other Cupriavidus strains by correspondence between the respective CHR1 replicons but also displays similarity to the genomes of more distantly related species as a result of gene transfer and through the presence of large genomic islands. The presence of at least 57 IS elements and 19 transposons and the ability to take in and express foreign genes indicates a very dynamic and complex genome shaped by evolutionary forces. The genome data show that C. metallidurans CH34 is particularly well equipped to live in extreme conditions and anthropogenic environments that are rich in metals.

Antimicrobial silver: Uses, toxicity and potential for resistance

This review gives a comprehensive overview of the widespread use and toxicity of silver compounds in many biological applications. Moreover, the bacterial silver resistance mechanisms and their spread in the environment are discussed. This study shows that it is important to understand in detail how silver and silver nanoparticles exert their toxicity and to understand how bacteria acquire silver resistance. Silver ions have shown to possess strong antimicrobial properties but cause no immediate and serious risk for human health, which led to an extensive use of silver-based products in many applications. However, the risk of silver nanoparticles is not yet clarified and their widespread use could increase silver release in the environment, which can have negative impacts on ecosystems. Moreover, it is shown that silver resistance determinants are widely spread among environmental and clinically relevant bacteria. These resistance determinants are often located on mobile genetic elements, facilitating their spread. Therefore, detailed knowledge of the silver toxicity and resistance mechanisms can improve its applications and lead to a better understanding of the impact on human health and ecosystems.

Inactivation of Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure

ABSTRACT We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens,Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except bothSalmonella species showed higher inactivation in the presence of 100 μg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coliand P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.

High pressure increases bactericidal activity and spectrum of lactoferrin, lactoferricin and nisin.

We have studied the inactivation of a panel of eight test bacteria (two Escherichia coli strains, Salmonella enteritidis, Salmonella typhimurium, Shigella sonnei, Shigella flexneri, Pseudomonas fluorescens and Staphylococcus aureus) by high pressure in the presence of bovine lactoferrin (500 microg/ml), pepsin hydrolysate of lactoferrin (500 microg/ml), lactoferricin (20 microg/ml) and nisin (100 IU/ml). None of these compounds, at the indicated dosage, were bactericidal when applied at atmospheric pressure, except nisin, which caused a low level of inactivation of the bacteria. Under high pressure, lactoferrin, lactoferrin hydrolysate and lactoferricin displayed bactericidal activity against some of the test bacteria, however, the former had a narrower bactericidal spectrum than the two latter compounds. The bactericidal efficiency and spectrum of nisin were also enhanced under high pressure. The sensitisation of the test bacteria to these antimicrobials under pressure was transient, since no bactericidal activity was observed when bacteria were pressure treated before exposure to the compounds. We propose a mechanism of pressure-promoted uptake of these antimicrobial proteins and peptides in gram-negative bacteria to explain this sensitisation.

An SOS Response Induced by High Pressure in Escherichia coli

Although pressure is an important environmental parameter in microbial niches such as the deep sea and is furthermore used in food preservation to inactivate microorganisms, the fundamental understanding of its effects on bacteria remains fragmentary. Our group recently initiated differential fluorescence induction screening to search for pressure-induced Escherichia coli promoters and has already reported induction of the heat shock regulon. Here the screening was continued, and we report for the first time that pressure induces a bona fide SOS response in E. coli, characterized by the RecA and LexA-dependent expression of uvrA, recA, and sulA. Moreover, it was shown that pressure is capable of triggering lambda prophage induction in E. coli lysogens. The remnant lambdoid e14 element, however, could not be induced by pressure, as opposed to UV irradiation, indicating subtle differences between the pressure-induced and the classical SOS response. Furthermore, the pressure-induced SOS response seems not to be initiated by DNA damage, since DeltarecA and lexA1 (Ind-) mutants, which are intrinsically hypersensitive to DNA damage, were not sensitized or were only very slightly sensitized for pressure-mediated killing and since pressure treatment was not found to be mutagenic. In light of these findings, the current knowledge of pressure-mediated effects on bacteria is discussed.

Use of the rotating wall vessel technology to study the effect of shear stress on growth behaviour of Pseudomonas aeruginosa PA01

The biofilm phenotype of Pseudomonas aeruginosa enables this opportunistic pathogen to develop resistance to the immune system and antimicrobial agents. Pseudomonas aeruginosa biofilms are generated under varying levels of shear stress, depending on the infection site. In the lung mucus of cystic fibrosis (CF) patients, P. aeruginosa forms matrix-enclosed microcolonies which cause chronic infections representing the major cause of mortality in CF patients. The lung mucus of CF patients is probably characterized by low fluid shear as the main shear-causing factor, i.e. mucociliary clearance, is absent. In this study, the influence of fluid shear on the growth behaviour of P. aeruginosa PA01 was investigated using a low-shear suspension culture device, the rotating wall vessel (RWV). Cultivation in low shear induced a self-aggregating phenotype of P. aeruginosa PA01, resulting in the formation of biofilms in suspension similar to what has been described in CF mucus. The addition of a ceramic bead to the culture medium in the RWV created a higher-shear condition which led to the formation of surface-attached rather than suspension biofilms. In low-shear culture conditions, a significant increase of the rhl N-butanoyl-l-homoserine lactone (C(4)-HSL) directed quorum sensing (QS) system, and the psl polysaccharide synthetic locus was demonstrated using gene expression analysis. Accordingly, the low-shear condition induced a higher production of rhamnolipids, which is controlled by the C(4)-HSL QS-system and is known to play a role in CF lung pathology. These results indicate that fluid shear has an impact on the growth phenotype of P. aeruginosa which might play a role in CF lung infections caused by this bacterium.

Response of Pseudomonas aeruginosa PAO1 to low shear modelled microgravity involves AlgU regulation.

As a ubiquitous environmental organism that is occasionally part of the human flora, Pseudomonas aeruginosa could pose a health hazard for the immunocompromised astronauts during long-term missions. Therefore, insights into the behaviour of P. aeruginosa under spaceflight conditions were gained using two spaceflight-analogue culture systems: the rotating wall vessel (RWV) and the random position machine (RPM). Microarray analysis of P. aeruginosa PAO1 grown in the low shear modelled microgravity (LSMMG) environment of the RWV, compared with the normal gravity control (NG), revealed an apparent regulatory role for the alternative sigma factor AlgU (RpoE-like). Accordingly, P. aeruginosa cultured in LSMMG exhibited increased alginate production and upregulation of AlgU-controlled transcripts, including those encoding stress-related proteins. The LSMMG increased heat and oxidative stress resistance and caused a decrease in the oxygen transfer rate of the culture. This study also showed the involvement of the RNA-binding protein Hfq in the LSMMG response, consistent with its previously identified role in the Salmonella LSMMG and spaceflight response. The global transcriptional response of P. aeruginosa grown in the RPM was highly similar to that in NG. Fluid mixing was assessed in both systems and is believed to be a pivotal factor contributing to transcriptional differences between RWV- and RPM-grown P. aeruginosa. This study represents the first step towards the identification of virulence mechanisms of P. aeruginosa activated in response to spaceflight-analogue conditions, and could direct future research regarding the risk assessment and prevention of Pseudomonas infections during spaceflight and in immunocompromised patients.

N-Acyl-l-Homoserine Lactone Quorum Sensing Controls Butanediol Fermentation in Serratia plymuthica RVH1 and Serratia marcescens MG1

Butanediol fermentation in two Serratia species is shown to be affected by N-acyl-L-homoserine lactone-dependent quorum sensing. Knockout of quorum-sensing signal production caused a shift towards enhanced acid production, resulting in early growth arrest, which was reversible by the addition of synthetic signal molecules.

Heavy metal resistance in Cupriavidus metallidurans CH34 is governed by an intricate transcriptional network

The soil bacterium Cupriavidus metallidurans CH34 contains a high number of heavy metal resistance genes making it an interesting model organism to study microbial responses to heavy metals. In this study the transcriptional response of strain CH34 was measured when challenged to sub-lethal concentrations of various essential or toxic metals. Based on the global transcriptional responses for each challenge and the overlap in upregulated genes between different metal responses, the sixteen metals were clustered in three groups. In addition, the transcriptional response of already known metal resistance genes was assessed, and new metal response gene clusters were identified. The majority of the studied metal response loci showed similar expression profiles when cells were exposed to different metals, suggesting complex interplay at transcriptional level between the different metal responses. The pronounced redundancy of these metal resistant regions—as illustrated by the large number of paralogous genes—combined with the phylogenetic distribution of these metal response regions within either evolutionary related or other metal resistant bacteria, provides important insights on the recent evolutionary forces shaping this naturally soil-dwelling bacterium into a highly metal-resistant strain well adapted to harsh and anthropogenic environments.

New mobile genetic elements in Cupriavidus metallidurans CH34, their possible roles and occurrence in other bacteria

Cupriavidus metallidurans strain CH34 is a β-Proteobacterium that thrives in low concentrations of heavy metals. The genetic determinants of resistance to heavy metals are located on its two chromosomes, and are particularly abundant in the two megaplasmids, pMOL28 and pMOL30. We explored the involvement of mobile genetic elements in acquiring these and others traits that might be advantageous in this strain using genome comparison of Cupriavidus/Ralstonia strains and related β-Proteobacteria. At least eleven genomic islands were identified on the main replicon, three on pMOL28 and two on pMOL30. Multiple islands contained genes for heavy metal resistance or other genetic determinants putatively responding to harsh environmental conditions. However, cryptic elements also were noted. New mobile genetic elements (or variations of known ones) were identified through synteny analysis, allowing the detection of mobile genetic elements outside the bias of a selectable marker. Tn4371-like conjugative transposons involved in chemolithotrophy and degradation of aromatic compounds were identified in strain CH34, while similar elements involved in heavy metal resistance were found in Delftia acidovorans SPH-1 and Bordetella petrii DSM12804. We defined new transposons, viz., Tn6048 putatively involved in the response to heavy metals and Tn6050 carrying accessory genes not classically associated with transposons. Syntenic analysis also revealed new transposons carrying metal response genes in Burkholderia xenovorans LB400, and other bacteria. Finally, other putative mobile elements, which were previously unnoticed but apparently common in several bacteria, were also revealed. This was the case for triads of tyrosine-based site-specific recombinases and for an int gene paired with a putative repressor and associated with chromate resistance.