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.

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High-throughput amplicon sequencing has become a well-established approach for microbial community profiling. Correlating shifts in the relative abundances of bacterial taxa with environmental gradients is the goal of many microbiome surveys. As the abundances generated by this technology are semi-quantitative by definition, the observed dynamics may not accurately reflect those of the actual taxon densities. We combined the sequencing approach (16S rRNA gene) with robust single-cell enumeration technologies (flow cytometry) to quantify the absolute taxon abundances. A detailed longitudinal analysis of the absolute abundances resulted in distinct abundance profiles that were less ambiguous and expressed in units that can be directly compared across studies. We further provide evidence that the enrichment of taxa (increase in relative abundance) does not necessarily relate to the outgrowth of taxa (increase in absolute abundance). Our results highlight that both relative and absolute abundances should be considered for a comprehensive biological interpretation of microbiome surveys. The ISME Journal (2017) 11, 584-587; doi:10.1038/ismej.2016 published online 9 September 2016 Recent advancements in high-throughput sequencing of marker genes, such as the 16S rRNA gene, have provided microbial ecologists the tools to accurately infer the relative composition of microbial communities (Franzosa et al., 2015). This resulted in a widespread application of the technology in longitudinal studies where shifts in community structure are related to environmental variables and functional outputs (Faust et al., 2015;Wilhelm et al., 2015). An inherent limitation of the sequencing technology is that the calculated taxon abundances comprise relative values (Widder et al., 2016). Hence, caution must be taken with the biological interpretation of these values, since inter-sample differences in cell density are not considered. To our knowledge, there are no descriptive studies that assess the extent to which relative abundances deliver a skewed image of the actual microbial community dynamics. In this study, we combined robust cell density measurements from flow cytometry (Prest et al., 2013;Van Nevel et al., 2013) with the relative abundances derived from 16S rRNA gene amplicon sequencing. We performed two extensive longitudinal surveys on the central water reservoir of a cooling water system. This engineered freshwater ecosystem was subjected to highly controlled operational phases (Supplementary Information and data set). We quantified the absolute taxon abundances and assessed whether additional insights could be attained with the combined approach.Based on the sample-specified total cell density, the absolute taxon abundances were calculated for each time point. Individual taxon densities ranged from 0.5 to 1 679 cells per μl. Several inter-taxon differences became apparent by performing ordinary least squares regression analysis between the relative and absolute abundances. We focused on the three most abundant taxa, which ...

Summary Measuring the microbial diversity in natural and engineered environments is important for ecosystem characterization, ecosystem monitoring and hypothesis testing. Although the conventional assessment through single marker gene surveys has resulted in major advances, the complete procedure remains slow (i.e. weeks to months), labour‐intensive and susceptible to multiple sources of laboratory and data processing bias. Growing interest, in highly resolved, temporal surveys of microbial diversity, necessitates rapid, inexpensive and robust analytical platforms that require limited computational effort. Here, we demonstrate that sensitive single‐cell measurements of phenotypic attributes, obtained via flow cytometry, can provide fast (i.e. within minutes) first‐line assessments of microbial diversity dynamics, without demanding extensive sample preparation and downstream data processing. We developed a data processing pipeline that fits bivariate kernel density functions to phenotypic parameter combinations of an entire microbial community and concatenates them to a single one‐dimensional phenotypic fingerprint. By calculating established diversity metrics from such phenotypic fingerprints, we construct an alternative interpretation of the microbial diversity that incorporates distinct phenotypic traits underlying cell‐to‐cell heterogeneity (i.e. morphology and nucleic acid content). Based on a detailed longitudinal study of a highly dynamic microbial ecosystem, our approach delivered temporal alpha diversity profiles that strongly correlated with the reference diversity, as estimated by 16S rRNA amplicon sequencing. This strongly suggests that the distribution of a limited amount of phenotypic features within a microbial community already provides sufficient resolving power for the measurement of diversity dynamics at the species level. We present a fast, robust analysis method for monitoring the microbial biodiversity of natural and engineered ecosystems that correlates well with the conventional marker gene surveys. Our work has both applied and fundamental implications that stretch from ecosystem monitoring and studies on microbial community dynamics, to supervised sampling strategies. Furthermore, our approach offers perspectives for the development of online and in situ monitoring systems for microbial ecosystems.

Assessing bacterial behavior in microgravity is important for risk assessment and prevention of infectious diseases during spaceflight missions. Furthermore, this research field allows the unveiling of novel connections between low-fluid-shear regions encountered by pathogens during their natural infection process and bacterial virulence. This study is the first to characterize the spaceflight-induced global transcriptional and proteomic responses of Pseudomonas aeruginosa, an opportunistic pathogen that is present in the space habitat. P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins, with Hfq as a global transcriptional regulator. Since Hfq was also differentially regulated in spaceflight-grown Salmonella enterica serovar Typhimurium, Hfq represents the first spaceflight-induced regulator acting across bacterial species. The major P. aeruginosa virulence-related genes induced in spaceflight were the lecA and lecB lectin genes and the gene for rhamnosyltransferase (rhlA), which is involved in rhamnolipid production. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data for this organism grown in microgravity analogue conditions using the rotating wall vessel (RWV) bioreactor. Interesting similarities were observed, including, among others, similarities with regard to Hfq regulation and oxygen metabolism. While RWV-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism, P. aeruginosa cultured in spaceflight presumably adopted an anaerobic mode of growth, in which denitrification was most prominent. Whether the observed changes in pathogenesis-related gene expression in response to spaceflight culture could lead to an alteration of virulence in P. aeruginosa remains to be determined and will be important for infectious disease risk assessment and prevention, both during spaceflight missions and for the general public.The microgravity environment associated with spaceflight is unique and has a profound effect on both host and pathogen cells, with potential implications for infectious disease. From the host point of view, astronauts experience a compromised immune response under spaceflight conditions, as reflected in cellular alterations of both the innate and adaptive immune systems (23,26,40). Spaceflight has been shown to alter the response of monocytes, isolated from astronauts preflight and in flight, to . Further, simulation of aspects of this microgravity-associated decreased immune response, using the hind limb unloaded mouse model, showed an enhanced susceptibility of these animals to bacterial infection (3,6). From the pathogen's perspective, bacterial obligate and opportunistic pathogens have been found to exhibit enhanced stress resistance phenotypes following growth under both true spaceflight and microgravity analogue conditions (13,30,33,(46)(47)(48)(49). In response to the spaceflight environment, global transcriptional and proteomic changes were observed for the ente...