Local Adaptation of Legionella pneumophila within a Hospital Hot Water System Increases Tolerance to Copper.

In large-building water systems, Legionella pneumophila is exposed to common environmental stressors such as copper. The aim of this study was to evaluate the susceptibility to copper of L. pneumophila isolates recovered from various sites: two clinical and seven environmental isolates from hot water system biofilm and water and from cooling tower water. After a 1-week acclimation in simulated drinking water, strains were exposed to various copper concentrations (0.8 to 5 mg/liter) for over 672 h. Complete loss of culturability was observed for three isolates following copper exposure to 5 mg/liter for 672 h. Two sequence type 1427 (ST1427)-like isolates were highly sensitive to copper, while the other two, isolated from biofilm samples, maintained higher culturability. The expression of the copper resistance gene copA evaluated by reverse transcription-quantitative PCR (RT-qPCR) was significantly higher for the biofilm isolates. All four ST1427-like isolates were recovered from the same water system during an outbreak. Whole-genome sequencing results confirmed that the four isolates are very close phylogenetically, differing by only 29 single nucleotide polymorphisms, suggesting in situ adaptation to microenvironmental conditions, possibly due to epigenetic regulation. These results indicate that the immediate environment within a building water distribution system influences the tolerance of L. pneumophila to copper. Increased contact of L. pneumophila biofilm strains with copper piping or copper alloys in the heat exchanger might lead to local adaptation. The phenotypic differences observed between water and biofilm isolates from the hot water system of a health care facility warrants further investigation to assess the relevance of evaluating disinfection performances based on water sampling alone.IMPORTANCELegionella pneumophila is a pathogen indigenous to natural and large building water systems in the bulk and the biofilm phases. The immediate environment within a system can impact the tolerance of L. pneumophila to environmental stressors, including copper. In health care facilities, copper levels in water can vary, depending on water quality, plumbing materials, and age. This study evaluated the impact of the isolation site (water versus biofilm, hot water system versus cooling tower) within building water systems. Closely related strains isolated from a health care facility hot water system exhibited variable tolerance to copper stress, shown by differential expression of copA, with biofilm isolates displaying highest expression and tolerance. Relying on the detection of L. pneumophila in water samples following exposure to environmental stressors such as copper may underestimate the prevalence of L. pneumophila, leading to inappropriate risk management strategies and increasing the risk of exposure for vulnerable patients.

Deletion of oxyR in Legionella pneumophila causes growth defect on agar.

The intracellular pathogen Legionella pneumophila (Lp) is a strict aerobe, surviving and replicating in environments where it frequently encounters reactive oxygen species (ROS), such as the nutrient-poor water environment and its replicative niche inside host cells. In many proteobacteria, the LysR-type regulator OxyR controls the oxidative stress response; however, the importance of the OxyR homologue in Lp is still unclear. Therefore, we undertook the characterization of phenotypes associated with the deletion of oxyR in Lp. Contrary to the wild type, the oxyR deletion mutant exhibits a severe growth defect on charcoal - yeast extract (CYE) agar lacking α-ketoglutarate supplementation. Growth in AYE broth (CYE without agar and charcoal), in amoeba and in human cultured macrophages, and survival in water is unaffected by the deletion. Supplementing CYE agar with antioxidants that neutralize ROS or introducing the oxyR gene in trans rescues the observed growth defect. Moreover, the mutant grows as well as the wild type on CYE plates made with agarose instead of agar, suggesting that a compound present in the latter is responsible for the growth defect phenotype.

Packaging of Campylobacter jejuni into Multilamellar Bodies by the Ciliate Tetrahymena pyriformis.

Campylobacter jejuniis the leading cause of bacterial gastroenteritis worldwide. Transmission to humans occurs through consumption of contaminated food or water. The conditions affecting the persistence of C. jejuniin the environment are poorly understood. Some protozoa package and excrete bacteria into multilamellar bodies (MLBs). Packaged bacteria are protected from deleterious conditions, which increases their survival. We hypothesized that C. jejuni could be packaged under aerobic conditions by the amoeba Acanthamoeba castellanii or the ciliate Tetrahymena pyriformis, both of which are able to package other pathogenic bacteria.A. castellanii did not produce MLBs containing C. jejuni In contrast, when incubated with T. pyriformis,C. jejuni was ingested, packaged in MLBs, and then expelled into the milieu. The viability of the bacteria inside MLBs was confirmed by microscopic analyses. The kinetics of C. jejuni culturability showed that packaging increased the survival of C. jejuniup to 60 h, in contrast to the strong survival defect seen in ciliate-free culture. This study suggests that T. pyriformis may increase the risk of persistence of C. jejuniin the environment and its possible transmission between different reservoirs in food and potable water through packaging.

Transcriptomic changes of Legionella pneumophila in water.

BACKGROUND: Legionella pneumophila (Lp) is a water-borne opportunistic pathogen. In water, Lp can survive for an extended period of time until it encounters a permissive host. Therefore, identifying genes that are required for survival in water may help develop strategies to prevent Legionella outbreaks. RESULTS: We compared the global transcriptomic response of Lp grown in a rich medium to that of Lp exposed to an artificial freshwater medium (Fraquil) for 2, 6 and 24 hours. We uncovered successive changes in gene expression required for the successful adaptation to a nutrient-limited water environment. The repression of major pathways involved in cell division, transcription and translation, suggests that Lp enters a quiescent state in water. The induction of flagella associated genes (flg, fli and mot), enhanced-entry genes (enh) and some Icm/Dot effector genes suggests that Lp is primed to invade a suitable host in response to water exposure. Moreover, many genes involved in resistance to antibiotic and oxidative stress were induced, suggesting that Lp may be more tolerant to these stresses in water. Indeed, Lp exposed to water is more resistant to erythromycin, gentamycin and kanamycin than Lp cultured in rich medium. In addition, the bdhA gene, involved in the degradation pathway of the intracellular energy storage compound polyhydroxybutyrate, is also highly expressed in water. Further characterization show that expression of bdhA during short-term water exposure is dependent upon RpoS, which is required for the survival of Lp in water. Deletion of bdhA reduces the survival of Lp in water at 37 °C. CONCLUSIONS: The increase of antibiotic resistance and the importance of bdhA to the survival of Lp in water seem consistent with the observed induction of these genes when Lp is exposed to water. Other genes that are highly induced upon exposure to water could also be necessary for Lp to maintain viability in the water environment.

A regulatory feedback loop between RpoS and SpoT supports the survival of Legionella pneumophila in water.

Legionella pneumophila is a waterborne pathogen, and survival in the aquatic environment is central to its transmission to humans. Therefore, identifying genes required for its survival in water could help prevent Legionnaires' disease outbreaks. In the present study, we investigate the role of the sigma factor RpoS in promoting survival in water, where L. pneumophila experiences severe nutrient deprivation. The rpoS mutant showed a strong survival defect compared to the wild-type strain in defined water medium. The transcriptome of the rpoS mutant during exposure to water revealed that RpoS represses genes associated with replication, translation, and transcription, suggesting that the mutant fails to shut down major metabolic programs. In addition, the rpoS mutant is transcriptionally more active than the wild-type strain after water exposure. This could be explained by a misregulation of the stringent response in the rpoS mutant. Indeed, the rpoS mutant shows an increased expression of spoT and a corresponding decrease in the level of (p)ppGpp, which is due to the presence of a negative feedback loop between RpoS and SpoT. Therefore, the lack of RpoS causes an aberrant regulation of the stringent response, which prevents the induction of a successful response to starvation.

Rapid and specific SPRi detection of L. pneumophila in complex environmental water samples.

Legionellosis is a very devastating disease worldwide mainly due to unpredictable outbreaks in man-made water systems. Developing a highly specific and sensitive rapid detection system that detects only metabolically active bacteria is a main priority for water quality assessment. We previously developed a versatile technique for sensitive and specific detection of synthetic RNA. In the present work, we further investigated the performance of the developed biosensor for detection of Legionella pneumophila in complex environmental samples, particularly those containing protozoa. The specificity and sensitivity of the detection system were verified using total RNA extracted from L. pneumophila in spiked water co-cultured with amoebae. We demonstrated that the expression level of ribosomal RNA (rRNA) is extremely dependent on the environmental conditions. The presence of amoebae with L. pneumophila, especially in nutrition-deprived samples, increased the amount of L. pneumophila 15-fold after 1 week as measured through the expression of 16s rRNA. Using the developed surface plasmon resonance imaging (SPRi) detection method, we were also able to successfully detect L. pneumophila within 3 h, both in the presence and absence of amoebae in the complex environmental samples obtained from a cooling water tower. These findings suggest that the developed biosensing system is a viable method for rapid, real-time and effective detection not only for L. pneumophila in environmental samples but also to assess the risk associated with the use of water contaminated with other pathogens.

Facets of small RNA-mediated regulation in Legionella pneumophila.

Legionella pneumophila is a water-borne pathogen that causes a severe lung infection in humans. It is able to replicate inside amoeba in the water environment, and inside lung macrophages in humans. Efficient regulation of gene expression is critical for responding to the conditions that L. pneumophila encounters and for intracellular multiplication in host cells. In the last two decades, many reports have contributed to our understanding of the critical importance of small regulatory RNAs (sRNAs) in the regulatory network of bacterial species. This report presents the current state of knowledge about the sRNAs expressed by L. pneumophila and discusses a few regulatory pathways in which sRNAs should be involved in this pathogen.

Assessment of monitoring approaches to control Legionella pneumophila within a complex cooling tower system.

Precise and rapid methods are needed to improve monitoring approaches of L. pneumophila (Lp) in cooling towers (CTs) to allow timely operational adjustments and prevent outbreaks. The performance of liquid culture (ASTM D8429-21) and an online qPCR device were first compared to conventional filter plate culture (ISO 11731-2017), qPCR and semi-automated qPCR at three spiked concentrations of Lp (serogroup 1) validated by flow cytometry (total/viable cell count). The most accurate was qPCR, followed by liquid culture, online and semi-automated qPCR, and lastly, by a significant margin, filter plate culture. An industrial CT system was monitored using liquid and direct plate culture by the facility, qPCR and online qPCR. Direct plate and liquid culture results agreed at regulatory sampling point, supporting the use of the faster liquid culture for monitoring culturable Lp. During initial operation, qPCR and online qPCR results were within one log of culture at the primary pump before deviating after first cleaning. Other points revealed high spatial variability of Lp. The secondary pumps and chiller had the most positivity and highest concentrations by both qPCR and liquid culture compared to the basin and infeed tank. Altogether, this suggests that results from monthly compliance sampling at a single location with plate culture are not representative of Lp risks in this CT due to the high temporal and spatial variability. The primary pump, rather than the CT basin, should be designated for sampling, as it is representative of the health risk. An annual multi point survey of the system should be conducted to identify and target Lp hot spots. Generally, a combination of liquid culture for compliance and frequent qPCR for process control provides a more agile and robust monitoring scheme than plate culture alone, enabling early treatment adjustments, due to lower limit of detection (LOD) and turnover time.

Analysis of the transcriptome of Legionella pneumophila hfq mutant reveals a new mobile genetic element.

Hfq is a small RNA-binding protein involved in the post-transcriptional regulation of gene expression by affecting the stability of the mRNA and by mediating efficient pairing between small regulatory RNAs and their target mRNAs. In Legionella pneumophila, the aetiological agent of Legionnaires' disease, mutation of hfq results in increased duration of the lag phase and reduced growth in low-iron medium. In an effort to uncover genes potentially regulated by Hfq, the transcriptome of an hfq mutant strain was compared to that of the wild-type. Unexpectedly, many genes located within a 100 kb genomic island, including a section of the previously identified efflux island, were overexpressed in the hfq mutant strain. Since this island contains a putative conjugative system and an integrase, it was postulated that it could be a new integrated mobile genetic element. PCR analysis revealed that this region exists both as an integrated and as an episomal form in the cell population and that it undergoes differential excision in the hfq mutant background, which was further confirmed by trans-complementation of the hfq mutation. This new plasmid-like element was named pLP100. Differential excision did not affect the copy number of pLP100 at the population level. This region contains a copper efflux pump encoded by copA, and increased resistance to copper was observed for the hfq mutant strain that was abrogated in the complemented strain. A strain carrying a mutation of hfq and a deletion of the right side recombination site, attR, showed that overexpression of pLP100 genes and increased copper resistance in the hfq mutant strain were dependent upon excision of pLP100.

Detection of Diverse Sequence Types of Legionella pneumophila by Legiolert Enzymatic-Based Assay and the Development of a Long-Term Storage Protocol.

Legiolert is a rapid culture-based enzymatic method for the detection and quantification of Legionella pneumophila in potable and nonpotable water samples. We aimed to assess the ability of this assay to detect diverse sequence types and validated a simple method to preserve samples. We used this assay on 253 potable and 165 nonpotable cooling tower water samples from various buildings in Québec, Canada, and performed sequence-based typing on 96 isolates. Six sequence types were identified, including ST1, ST378, ST1427, ST2859, ST3054, and ST3069. Whole-genome sequencing revealed that ST2859 was a member of the L. pneumophila subspecies fraseri. Additional tests with pure isolates also found that subspecies Pascullei and Raphaeli could be detected via Legiolert. Eight storage methods, including the current recommendation to store Legiolert trays at 4°C, were evaluated for their ability to preserve viable cultures. Of those, storage of Legiolert culture with 10% glycerol at -80°C produced the best results, fully preserving culturable Legionella for at least 12.5 months. We incorporated these findings into a standard procedure for processing Legiolert packets. Overall, Legiolert captures a variety of common and new STs in addition to important L. pneumophila subspecies and can be easily stored, which allows the conservation of a population of isolates for later characterization. IMPORTANCE Legionnaires' disease is caused by the bacterium Legionella pneumophila, which can be found in a variety of water systems. When outbreaks of Legionnaires' disease occur, it is necessary to find the water systems transmitting the bacterium to humans. Access to historical isolates from water system samples is key for success in identifying sources but current regulations and isolation protocols mean very few isolates are obtained and stored long-term. We showed here that the Legiolert test could detect and produce isolates of a variety of L. pneumophila subspecies and types. In addition, the Legiolert test medium containing a representative population of isolates could be preserved for at least 12 months at -80°C with the addition of glycerol to the test medium. Therefore, we confirmed that the Legiolert method could be a useful tool for retrospective analysis of potential sources for an outbreak.

Impact of Stagnation on the Diversity of Cyanobacteria in Drinking Water Treatment Plant Sludge.

Health-related concerns about cyanobacteria-laden sludge of drinking water treatment plants (DWTPs) have been raised in the past few years. Microscopic taxonomy, shotgun metagenomic sequencing, and microcystin (MC) measurement were applied to study the fate of cyanobacteria and cyanotoxins after controlled sludge storage (stagnation) in the dark in a full-scale drinking water treatment plant within 7 to 38 days. For four out of eight dates, cyanobacterial cell growth was observed by total taxonomic cell counts during sludge stagnation. The highest observed cell growth was 96% after 16 days of stagnation. Cell growth was dominated by potential MC producers such as Microcystis, Aphanocapsa, Chroococcus, and Dolichospermum. Shotgun metagenomic sequencing unveiled that stagnation stress shifts the cyanobacterial communities from the stress-sensitive Nostocales (e.g., Dolichospermum) order towards less compromised orders and potential MC producers such as Chroococcales (e.g., Microcystis) and Synechococcales (e.g., Synechococcus). The relative increase of cyanotoxin producers presents a health challenge when the supernatant of the stored sludge is recycled to the head of the DWTP or discharged into the source. These findings emphasize the importance of a strategy to manage cyanobacteria-laden sludge and suggest practical approaches should be adopted to control health/environmental impacts of cyanobacteria and cyanotoxins in sludge.

Shotgun Metagenomic Sequencing to Assess Cyanobacterial Community Composition following Coagulation of Cyanobacterial Blooms.

The excessive proliferation of cyanobacteria in surface waters is a widespread problem worldwide, leading to the contamination of drinking water sources. Short- and long-term solutions for managing cyanobacterial blooms are needed for drinking water supplies. The goal of this research was to investigate the cyanobacteria community composition using shotgun metagenomics in a short term, in situ mesocosm experiment of two lakes following their coagulation with ferric sulfate (Fe2(SO4)3) as an option for source water treatment. Among the nutrient paramenters, dissolved nitrogen was related to Microcystis in both Missisquoi Bay and Petit Lac St. François, while the presence of Synechococcus was related to total nitrogen, dissolved nitrogen, dissolved organic carbon, and dissolved phosphorus. Results from the shotgun metagenomic sequencing showed that Dolichospermum and Microcystis were the dominant genera in all of the mesocosms in the beginning of the sampling period in Missisquoi Bay and Petit Lac St. François, respectively. Potentially toxigenic genera such as Microcystis were correlated with intracellular microcystin concentrations. A principal component analysis showed that there was a change of the cyanobacterial composition at the genus level in the mesocosms after two days, which varied across the studied sites and sampling time. The cyanobacterial community richness and diversity did not change significantly after its coagulation by Fe2(SO4)3 in all of the mesocosms at either site. The use of Fe2(SO4)3 for an onsite source water treatment should consider its impact on cyanobacterial community structure and the reduction of toxin concentrations.

Characterization of heterotrophic prokaryote subgroups in the Sfax coastal solar salterns by combining flow cytometry cell sorting and phylogenetic analysis.

Here, we combined flow cytometry (FCM) and phylogenetic analyses after cell sorting to characterize the dominant groups of the prokaryotic assemblages inhabiting two ponds of increasing salinity: a crystallizer pond (TS) with a salinity of 390 g/L, and the non-crystallizer pond (M1) with a salinity of 200 g/L retrieved from the solar saltern of Sfax in Tunisia. As expected, FCM analysis enabled the resolution of high nucleic acid content (HNA) and low nucleic acid content (LNA) prokaryotes. Next, we performed a taxonomic analysis of the bacterial and archaeal communities comprising the two most populated clusters by phylogenetic analyses of 16S rRNA gene clone library. We show for the first time that the presence of HNA and LNA content cells could also be extended to the archaeal populations. Archaea were detected in all M1 and TS samples, whereas representatives of Bacteria were detected only in LNA for M1 and HNA for TS. Although most of the archaeal sequences remained undetermined, other clones were most frequently affiliated to Haloquadratum and Halorubrum. In contrast, most bacterial clones belonged to the Alphaproteobacteria class (Phyllobacterium genus) in M1 samples and to the Bacteroidetes phylum (Sphingobacteria and Salinibacter genus) in TS samples.

Survival of extremely and moderately halophilic isolates of Tunisian solar salterns after UV-B or oxidative stress.

Adaptation to a solar saltern environment requires mechanisms providing tolerance not only to salinity but also to UV radiation (UVR) and to reactive oxygen species (ROS). We cultivated prokaryote halophiles from two different salinity ponds: the concentrator M1 pond (240 g·L(-1) NaCl) and the crystallizer TS pond (380 g·L(-1) NaCl). We then estimated UV-B and hydrogen peroxide resistance according to the optimal salt concentration for growth of the isolates. We observed a higher biodiversity of bacterial isolates in M1 than in TS. All strains isolated from TS appeared to be extremely halophilic Archaea from the genus Halorubrum. Culturable strains isolated from M1 included extremely halophilic Archaea (genera Haloferax, Halobacterium, Haloterrigena, and Halorubrum) and moderately halophilic Bacteria (genera Halovibrio and Salicola). We also found that archaeal strains were more resistant than bacterial strains to exposure to ROS and UV-B. All organisms tested were more resistant to UV-B exposure at the optimum NaCl concentration for their growth, which is not always the case for H(2)O(2). Finally, if these results are extended to other prokaryotes present in a solar saltern, we could speculate that UVR has greater impact than ROS on the control of prokaryote biodiversity in a solar saltern.

The Effects of Ferric Sulfate (Fe2(SO4)3) on the Removal of Cyanobacteria and Cyanotoxins: A Mesocosm Experiment.

Cyanobacterial blooms are a global concern. Chemical coagulants are used in water treatment to remove contaminants from the water column and could potentially be used in lakes and reservoirs. The aims of this study was to: 1) assess the efficiency of ferric sulfate (Fe2(SO4)3) coagulant in removing harmful cyanobacterial cells from lake water with cyanobacterial blooms on a short time scale, 2) determine whether some species of cyanobacteria can be selectively removed, and 3) determine the differential impact of coagulants on intra- and extra-cellular toxins. Our main results are: (i) more than 96% and 51% of total cyanobacterial cells were removed in mesocosms with applied doses of 35 mgFe/L and 20 mgFe/L, respectively. Significant differences in removing total cyanobacterial cells and several dominant cyanobacteria species were observed between the two applied doses; (ii) twelve microcystins, anatotoxin-a (ANA-a), cylindrospermopsin (CYN), anabaenopeptin A (APA) and anabaenopeptin B (APB) were identified. Ferric sulfate effectively removed the total intracellular microcystins (greater than 97% for both applied doses). Significant removal of extracellular toxins was not observed after coagulation with both doses. Indeed, the occasional increase in extracellular toxin concentration may be related to cells lysis during the coagulation process. No significant differential impact of dosages on intra- and extra-cellular toxin removal was observed which could be relevant to source water applications where optimal dosing is difficult to achieve.

Can Cyanobacterial Diversity in the Source Predict the Diversity in Sludge and the Risk of Toxin Release in a Drinking Water Treatment Plant?

Conventional processes (coagulation, flocculation, sedimentation, and filtration) are widely used in drinking water treatment plants and are considered a good treatment strategy to eliminate cyanobacterial cells and cell-bound cyanotoxins. The diversity of cyanobacteria was investigated using taxonomic cell counts and shotgun metagenomics over two seasons in a drinking water treatment plant before, during, and after the bloom. Changes in the community structure over time at the phylum, genus, and species levels were monitored in samples retrieved from raw water (RW), sludge in the holding tank (ST), and sludge supernatant (SST). Aphanothece clathrata brevis, Microcystis aeruginosa, Dolichospermum spiroides , and Chroococcus minimus were predominant species detected in RW by taxonomic cell counts. Shotgun metagenomics revealed that Proteobacteria was the predominant phylum in RW before and after the cyanobacterial bloom. Taxonomic cell counts and shotgun metagenomic showed that the Dolichospermum bloom occurred inside the plant. Cyanobacteria and Bacteroidetes were the major bacterial phyla during the bloom. Shotgun metagenomics also showed that Synechococcus, Microcystis , and Dolichospermum were the predominant detected cyanobacterial genera in the samples. Conventional treatment removed more than 92% of cyanobacterial cells but led to cell accumulation in the sludge up to 31 times more than in the RW influx. Coagulation/sedimentation selectively removed more than 96% of Microcystis and Dolichospermum. Cyanobacterial community in the sludge varied from raw water to sludge during sludge storage (1-13 days). This variation was due to the selective removal of coagulation/sedimentation as well as the accumulation of captured cells over the period of storage time. However, the prediction of the cyanobacterial community composition in the SST remained a challenge. Among nutrient parameters, orthophosphate availability was related to community profile in RW samples, whereas communities in ST were influenced by total nitrogen, Kjeldahl nitrogen (N- Kjeldahl), total and particulate phosphorous, and total organic carbon (TOC). No trend was observed on the impact of nutrients on SST communities. This study profiled new health-related, environmental, and technical challenges for the production of drinking water due to the complex fate of cyanobacteria in cyanobacteria-laden sludge and supernatant.

Diversity Assessment of Toxic Cyanobacterial Blooms during Oxidation.

Fresh-water sources of drinking water are experiencing toxic cyanobacterial blooms more frequently. Chemical oxidation is a common approach to treat cyanobacteria and their toxins. This study systematically investigates the bacterial/cyanobacterial community following chemical oxidation (Cl2, KMnO4, O3, H2O2) using high throughput sequencing. Raw water results from high throughput sequencing show that Proteobacteria, Actinobacteria, Cyanobacteria and Bacteroidetes were the most abundant phyla. Dolichospermum, Synechococcus, Microcystis and Nostoc were the most dominant genera. In terms of species, Dolichospermum sp.90 and Microcystis aeruginosa were the most abundant species at the beginning and end of the sampling, respectively. A comparison between the results of high throughput sequencing and taxonomic cell counts highlighted the robustness of high throughput sequencing to thoroughly reveal a wide diversity of bacterial and cyanobacterial communities. Principal component analysis of the oxidation samples results showed a progressive shift in the composition of bacterial/cyanobacterial communities following soft-chlorination with increasing common exposure units (CTs) (0-3.8 mg·min/L). Close cyanobacterial community composition (Dolichospermum dominant genus) was observed following low chlorine and mid-KMnO4 (287.7 mg·min/L) exposure. Our results showed that some toxin producing species may persist after oxidation whether they were dominant species or not. Relative persistence of Dolichospermum sp.90 was observed following soft-chlorination (0.2-0.6 mg/L) and permanganate (5 mg/L) oxidation with increasing oxidant exposure. Pre-oxidation using H2O2 (10 mg/L and one day contact time) caused a clear decrease in the relative abundance of all the taxa and some species including the toxin producing taxa. These observations suggest selectivity of H2O2 to provide an efficient barrier against toxin producing cyanobacteria entering a water treatment plant.

RskA Is a Dual Function Activator-Inhibitor That Controls SigK Activity Across Distinct Bacterial Genera.

It has been previously shown that RskA, the anti-Sigma factor K of Mycobacterium tuberculosis, inhibits SigK and that mutations in RskA promote high expression of the SigK regulon. The latter observation led us to hypothesize that RskA mutations lead to loss of the anti-Sigma factor function. In this report, we used natural and artificial mutations in RskA to determine the basis of the SigK-RskA partnership. Consistent with predictions, the N-terminal cytoplasmic portion of RskA was sufficient on its own to inhibit SigK. Unexpectedly, RskA also served as an activator of SigK. This activation depended on the same N-terminal region and was enhanced by the membrane-extracellular portion of RskA. Based on this, we engineered similar truncations in a Gram-negative bacterium, namely Yersinia enterocolitica. Again, we observed that, with specific alterations of RskA, we were able to enhance SigK activity. Together these results support an alternative mechanism of anti-Sigma factor function, that we could term modulator (activator-inhibitor) in both Actinobacteria and Gram-negative bacteria, suggesting that Sigma factor activation by anti-Sigma factors could be under-recognized.

Evidence of the importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa protein cytolytic activity in Escherichia coli.

Cyt1Aa is a cytolytic toxin, found together with the delta-endotoxins in Bacillus thuringiensis subsp. israelensis parasporal insecticidal crystals. The latter are used as an environmental friendly insecticide against mosquitoes and black flies. Contrary to Cry delta-endotoxin, the mode of action of Cyt1Aa is not completely understood. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1Aa gene named cyt1A97 has been isolated from a B. thuringiensis israelensis strain named BUPM97. The nucleotide sequence predicted a protein of 249 amino acids residues with a calculated molecular mass of 27 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix alpha C corresponding to a substitution of Met(115) by a Thr. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in Escherichia coli. It revealed that the mutated Cyt1A97 protein was over produced as inclusion bodies showing a very weak toxicity to E. coli contrarily to Cyt1A98 that stopped E. coli growth. Hence, hydrophobic residue Met at position 115 of Cyt1Aa should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa.