Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock.

Ribosome biogenesis is a complex and multistep process that depends on various assembly factors. To understand this process and identify the ribosome assembly intermediates, most studies have set out to delete or deplete these assembly factors. Instead, we took advantage of the impact of heat stress (45 °C) on the late stages of the biogenesis of the 30S ribosomal subunit to explore authentic precursors. Under these conditions, reduced levels of the DnaK chaperone proteins devoted to ribosome assembly lead to the transient accumulation of 21S ribosomal particles, which are 30S precursors. We constructed strains with different affinity tags on one early and one late 30S ribosomal protein and purified the 21S particles that form under heat shock. A combination of relative quantification using mass spectrometry-based proteomics and cryo-electron microscopy (cryo-EM) was then used to determine their protein contents and structures.

Synthesis and evaluation of 1,3,4-oxadiazole derivatives for development as broad-spectrum antibiotics.

The reality and intensity of antibiotic resistance in pathogenic bacteria calls for the rapid development of new antimicrobial drugs. In bacteria, trans-translation is the primary quality control mechanism for rescuing ribosomes arrested during translation. Because trans-translation is absent in eukaryotes but necessary to avoid ribosomal stalling and therefore essential for bacterial survival, it is a promising target either for novel antibiotics or for improving the activities of the protein synthesis inhibitors already in use. Oxadiazole derivatives display strong bactericidal activity against a large number of bacteria, but their effects on trans-translation were recently questioned. In this work, a series of new 1,3,4-oxadiazole derivatives and analogs were synthesized and assessed for their efficiency as antimicrobial agents against a wide range of gram-positive and gram-negative pathogenic strains. Despite the strong antimicrobial activity observed in these molecules, it turns out that they do not target trans-translation in vivo, but they definitely act on other cellular pathways.

Characterization of HicAB toxin-antitoxin module of Sinorhizobium meliloti.

BACKGROUND: Toxin-antitoxin (TA) systems are little genetic units generally composed of two genes encoding antitoxin and toxin. These systems are known to be involved in many functions that can lead to growth arrest and cell death. Among the different types of TA systems, the type II gathers together systems where the antitoxin directly binds and inhibits the toxin. Among these type II TA systems, the HicAB module is widely distributed in free-living Bacteria and Archaea and the toxin HicA functions via RNA binding and cleavage. The genome of the symbiotic Sinorhizobium meliloti encodes numerous TA systems and only a few of them are functional. Among the predicted TA systems, there is one homologous to HicAB modules. RESULTS: In this study, we characterize the HicAB toxin-antitoxin module of S. meliloti. The production of the HicA of S. meliloti in Escherichia coli cells abolishes growth and decreases cell viability. We show that expression of the HicB of S. meliloti counteracts HicA toxicity. The results of double hybrid assays and co-purification experiments allow demonstrating the interaction of HicB with the toxin HicA. Purified HicA, but not HicAB complex, is able to degrade ribosomal RNA in vitro. The analysis of separated domains of HicB protein permits us to define the antitoxin activity and the operator-binding domain. CONCLUSIONS: This study points out the first characterization of the HicAB system of the symbiotic S. meliloti whereas HicA is a toxin with ribonuclease activity and HicB has two domains: the COOH-terminal one that binds the operator and the NH2-terminal one that inhibits the toxin.

A Genetic Tool to Quantify trans-Translation Activity in Vivo.

In bacteria, trans-translation is the main quality control mechanism for rescuing ribosomes arrested during translation. This key process is universally conserved and plays a critical role in the viability and virulence of many pathogens. We developed a reliable in vivo double-fluorescence reporter system for the simultaneous quantification of both trans-translation and the associated proteolysis activities in bacteria. The assay was validated using mutant bacteria lacking tmRNA, SmpB, and the ClpP protease. Both antisense tmRNA-binding RNA and a peptide mimicking the SmpB C-terminal tail proved to be potent inhibitors of trans-translation in vivo. The double-fluorescent reporter was also tested with KKL-35, an oxadiazole derivative that is supposed to be a promising trans-translation inhibitor, and it surprisingly turns out that trans-translation is not the only target of KKL-35 in vivo.

Bacterial Community Diversity Harboured by Interacting Species.

All animals are infected by microbial partners that can be passengers or residents and influence many biological traits of their hosts. Even if important factors that structure the composition and abundance of microbial communities within and among host individuals have been recently described, such as diet, developmental stage or phylogeny, few studies have conducted cross-taxonomic comparisons, especially on host species related by trophic relationships. Here, we describe and compare the microbial communities associated with the cabbage root fly Delia radicum and its three major parasitoids: the two staphylinid beetles Aleochara bilineata and A. bipustulata and the hymenopteran parasitoid Trybliographa rapae. For each species, two populations from Western France were sampled and microbial communities were described through culture independent methods (454 pyrosequencing). Each sample harbored at least 59 to 261 different bacterial phylotypes but was strongly dominated by one or two. Microbial communities differed markedly in terms of composition and abundance, being mainly influenced by phylogenetic proximity but also geography to a minor extent. Surprisingly, despite their strong trophic interaction, parasitoids shared a very low proportion of microbial partners with their insect host. Three vertically transmitted symbionts from the genus Wolbachia, Rickettsia, and Spiroplasma were found in this study. Among them, Wolbachia and Spiroplasma were found in both the cabbage fly and at least one of its parasitoids, which could result from horizontal transfers through trophic interactions. Phylogenetic analysis showed that this hypothesis may explain some but not all cases. More work is needed to understand the dynamics of symbiotic associations within trophic network and the effect of these bacterial communities on the fitness of their hosts.

Visualizing compaction of polysomes in bacteria.

During protein synthesis, many translating ribosomes are bound together with an mRNA molecule to form polysomes (or polyribosomes). While the spatial organization of bacterial polysomes has been well studied in vitro, little is known about how they cluster when cellular conditions are highly constrained. To better understand this, we used electron tomography, template matching, and three-dimensional modeling to analyze the supramolecular network of ribosomes after induction of translational pauses. In Escherichia coli, we overexpressed an mRNA carrying a polyproline motif known to induce pausing during translation. When working with a strain lacking transfer-messenger RNA, the principle actor in the "trans-translation" rescuing system, the cells survived the hijacking of the translation machinery but this resulted in a sharp modification of the ribosomal network. The results of our experiments demonstrate that single ribosomes are replaced with large amounts of compacted polysomes. These polysomes are highly organized, principally forming hairpins and dimers of hairpins that stack together. We propose that these spatial arrangements help maintain translation efficiency when the rescue systems are absent or overwhelmed.

Inactivation of the LysR regulator Cj1000 of Campylobacter jejuni affects host colonization and respiration.

Transcriptional regulation mediates adaptation of pathogens to environmental stimuli and is important for host colonization. The Campylobacter jejuni genome sequence reveals a surprisingly small set of regulators, mostly of unknown function, suggesting an intricate regulatory network. Interestingly, C. jejuni lacks the homologues of ubiquitous regulators involved in stress response found in many other Gram-negative bacteria. Nonetheless, cj1000 is predicted to encode the sole LysR-type regulator in the C. jejuni genome, and thus may be involved in major adaptation pathways. A cj1000 mutant strain was constructed and found to be attenuated in its ability to colonize 1-day-old chicks. Complementation of the cj1000 mutation restored the colonization ability to wild-type levels. The mutant strain was also outcompeted in a competitive colonization assay of the piglet intestine. Oxygraphy was carried out for what is believed to be the first time with the Oroboros Oxygraph-2k on C. jejuni and revealed a role for Cj1000 in controlling O2 consumption. Furthermore, microarray analysis of the cj1000 mutant revealed both direct and indirect regulatory targets, including genes involved in energy metabolism and oxidative stress defences. These results highlight the importance of Cj1000 regulation in host colonization and in major physiological pathways.

Antimicrobial activities of isothiocyanates against Campylobacter jejuni isolates.

Food-borne human infection with Campylobacter jejuni is a medical concern in both industrialized and developing countries. Efficient eradication of C. jejuni reservoirs within live animals and processed foods is limited by the development of antimicrobial resistances and by practical problems related to the use of conventional antibiotics in food processes. We have investigated the bacteriostatic and bactericidal activities of two phytochemicals, allyl-isothiocyanate (AITC), and benzyl isothiocyanate (BITC), against 24 C. jejuni isolates from chicken feces, human infections, and contaminated foods, as well as two reference strains NCTC11168 and 81-176. AITC and BITC displayed a potent antibacterial activity against C. jejuni. BITC showed a higher overall antibacterial effect (MIC of 1.25-5 µg mL(-1)) compared to AITC (MIC of 50-200 µg mL(-1)). Both compounds are bactericidal rather than bacteriostatic. The sensitivity levels of C. jejuni isolates against isothiocyanates were neither correlated with the presence of a GGT (γ-Glutamyl Transpeptidase) encoding gene in the genome, with antibiotic resistance nor with the origin of the biological sample. However the ggt mutant of C. jejuni 81-176 displayed a decreased survival rate compared to wild-type when exposed to ITC. This work determined the MIC of two ITC against a panel of C. jejuni isolates, showed that both compounds are bactericidal rather than bacteriostatic, and highlighted the role of GGT enzyme in the survival rate of C. jejuni exposed to ITC.

Fitness of macrolide resistant Campylobacter coli and Campylobacter jejuni.

The aim of this study was to investigate the fitness of macrolide resistant Campylobacter coli and Campylobacter jejuni. The in vitro growth, the survival on food matrix, and the in vivo colonization of C. jejuni and C. coli susceptible isolates and their isogenic resistant mutants were studied. In vitro experiments demonstrated that macrolide resistance imposed a fitness cost when the susceptible strains and their isogenic resistant mutants were cultured in competition. When inoculated in food matrix, the resistant C. jejuni mutant was no longer detectable after 3 to 5 days but the susceptible strain remained detectable for over 18 days. No difference in survival in food matrix was observed between susceptible and resistant C. coli. When inoculated in vivo in chickens, the macrolide susceptible and resistant C. coli displayed similar levels of colonization, both in separated inoculations and during competitive assays. Strikingly, when mono-inoculated or co-inoculated into chickens, macrolide susceptible C. jejuni outcompeted the macrolide resistant population. However, a spontaneous mutant that evolved in vivo showed a colonization capacity similar to the susceptible strain. Our findings demonstrate the effect of macrolide resistance on the fitness of Campylobacter but suggest that evolved mutants may be as fit as susceptible strains.

Inhibition of adhesion of uropathogenic Escherichia coli bacteria to uroepithelial cells by extracts from cranberry.

Cranberry extract has been reported as a therapeutic agent, mainly in urinary tract infections due to its anti-adhesive capacity. In order to compare the effects of proanthocyanidin (procyanidin) (PAC)-standardized cranberry extracts and commercial PAC A2, we first investigated the presence of genes encoding known adhesins on 13 strains of uropathogenic strains coming from patients with cystisis. After this characterization, the anti-adhesive effects of PAC A2 were assayed on selected uropathogenic Escherichia coli strains before testing cranberry extracts. Before checking inhibitory effect on bacterial adhesion to cells, we showed that neither PAC A2 or three cranberry extracts (A, B, and C) specifically inhibited the growth and did not supply any potential nutrient to E. coli strains, including the unrelated control strain. PAC A2 exhibited an inhibitory effect on the adhesion of two selected uropathogenic strains of E. coli. This work also showed that a preliminary exposure of bacteria to PAC A2 significantly reduced the adhesion. This phenomenon has been also observed with a lesser impact when uroepithelial cells were pretreated with PAC A2. Moreover, the assays were more robust when bacteria were in fast growing conditions (exponential phase): the adhesion to uroepithelial cells was greater. Significant reduction of adhesion to urepithelial cells was observed: around 80% of inhibition of adhesion with the cranberry extracts at equivalent PAC concentration of 50 µg/mL. The effects of the different assayed extracts were not obviously different except for extract B, which inhibited approximately 55% of adhesion at an equivalent PAC concentration of 5 µg/mL.

MCLMAN, a new minimal medium for Campylobacter jejuni NCTC 11168.

The design of a defined synthetic minimal medium for Campylobacter jejuni strain NCTC 11168 that includes only inorganic salts and necessary amino acids and vitamins is useful in physiological assays and responses to exogeneous agents. In silico genomic analysis of biosynthesis pathways was preliminarily performed prior to experimental assays to determine (i) amino acids and vitamins necessary for improving the growth of C. jejuni strains, and (ii) the most appropriate sources of carbon, nitrogen and sulfur. The different sources of carbon, nitrogen and sulfur were analyzed by comparing growth parameters. A new minimal medium that contains inorganic salts, the amino acids l-cysteine, l-leucine, l-methionine and l-aspartic acid (nitrogen source), the vitamin niacinamide and lactate as a carbon source, named MCLMAN (medium cysteine leucine methionine aspartic acid niacinamide), was checked on some C. jejuni strains and showed similar growth ratios and final biomass when compared to the most frequently used medium, MEM (modified Eagle's medium), primarily designed for eukaryote cell culture and more complex than MCLMAN. Our results show that C. jejuni presents auxotrophy for cysteine and methionine and can be inhibited by ammonium sulfate. A simple minimal medium containing few amino acids and vitamins will facilitate physiological studies of different functions in C. jejuni strains submitted to different stresses.

RpoE2 of Sinorhizobium meliloti is necessary for trehalose synthesis and growth in hyperosmotic media.

Adaptation to osmotic stress can be achieved by the accumulation of compatible solutes that aid in turgor maintenance and macromolecule stabilization. The genetic regulation of solute accumulation is poorly understood, and has been described well at the molecular level only in enterobacteria. In this study, we show the importance of the alternative sigma factor RpoE2 in Sinorhizobium meliloti osmoadaptation. Construction and characterization of an S. meliloti rpoE2 mutant revealed compromised growth in hyperosmotic media. This defect was due to the lack of trehalose, a minor carbohydrate osmolyte normally produced in the initial stages of growth and in stationary phase. We demonstrate here that all three trehalose synthesis pathways are RpoE2 dependent, but only the OtsA pathway is important for osmoinducible trehalose synthesis. Furthermore, we confirm that the absence of RpoE2-dependent induction of otsA is the cause of the osmotic phenotype of the rpoE2 mutant. In conclusion, we have highlighted that, despite its low level, trehalose is a crucial compatible solute in S. meliloti, and the OtsA pathway induced by RpoE2 is needed for its accumulation under hyperosmotic conditions.

Detection of Listeria monocytogenes in raw and pasteurized liquid whole eggs and characterization by PFGE.

Listeria monocytogenes has been recognized as a human pathogen for decades and is known to be an important foodborne pathogen. There have been no documented foodborne L. monocytogenes illnesses due to the consumption of eggs or egg products, even though the bacterium has been isolated from faeces, body fluid, and oviducts of asymptomatic laying hens. In order to describe L. monocytogenes contamination of egg products, 144 liquid whole egg samples were collected from 3 different egg-breaking plants during 3 sampling periods. L. monocytogenes detection was performed on raw samples stored at 2 degrees C for two days (D+2) and on pasteurized samples stored at 2 degrees C at D+2 and at shelf-life date (SLD). L. monocytogenes was detected in 25 of the 144 raw egg samples collected, in 4 of the 144 pasteurized egg samples at D+2 and in 2 of the 144 ones analysed at SLD. Contamination of raw egg products appeared to be season dependant and was higher during summer and winter than during autumn. One hundred and ninety-six L. monocytogenes isolates were collected and serotyped; 3 serovars were demonstrated. The dominant serovar was L. monocytogenes 1/2a which was presented by 94.4% of the isolates. Typing of 196 L. monocytogenes isolates was carried out by macrorestriction of the genomic DNA with ApaI and AscI enzymes followed by pulsed field gel electrophoresis (PFGE). A large diversity was observed with 21 genotypes of L. monocytogenes, even for a given manufacturer. Nevertheless, most of the egg product samples were contaminated by one genotype, except for five samples which were contaminated by two or three distinct genotypes. The genotypes seem to be specific to each manufacturer. No cluster of L. monocytogenes was found to recur in the different plants over successive seasons.

Use of pulsed-field gel electrophoresis to characterize the heterogeneity and clonality of Salmonella serotype Enteritidis, Typhimurium and Infantis isolates obtained from whole liquid eggs.

Salmonella is a well-documented pathogen known to occur in a wide range of foods, especially poultry products. The most frequently reported food-sources of human infection are eggs and egg products. In this study, in order to describe Salmonella contamination of egg products, 144 liquid egg samples were collected from 3 different egg-breaking plants during the 3 sampling periods. Salmonella detection was performed on raw samples stored at 2 degrees C for 2 days (D+2) and on pasteurised samples stored at 2 degrees C at D+2 and at shelf-life date. Salmonella was detected in 130 of the 144 raw egg samples collected and in 11 of the 288 pasteurised egg samples analysed. 740 Salmonella isolates were collected and serotyped: 14 serovars were demonstrated. A great diversity, particularly during summer, was noted. The dominant serovars were S. Enteritidis, S. Typhimurium and S. Infantis, mainly found in whole raw egg products. Typing of 325 isolates of S. Enteritidis, 54 isolates of S. Typhimurium and 58 isolates of S. Infantis was carried out by macrorestriction of the genomic DNA with XbaI and SpeI enzymes followed by pulsed field gel electrophoresis (PFGE). The Salmonella Enteritidis isolates could be grouped into 3 clusters. Cluster 1 was predominant at all 3 egg-breaking companies during the different sampling periods. This cluster seemed to be adapted to the egg-breaking plants. Cluster 2 was linked to plant 1 and cluster 3 to plant 3. Two main clusters of Salmonella Typhimurium were demonstrated. Cluster A was mainly found at plant 2 during autumn. Plant 3 was contaminated by all the Salmonella Typhimurium genotypes but in a more sporadic manner during the three seasons studied. Plant 1 seemed to be less contaminated by Salmonella Typhimurium than the others. Three clusters and 2 genotypes of Salmonella Infantis were shown. The main cluster, cluster alpha, consisted of 75% of the S. Infantis isolates and was mainly found during summer at plants 1 and 3. Plant 2 seemed to be less contaminated by S. Infantis. In this study, molecular typing demonstrated that, although certain clusters were common to all three companies, specific clusters, notably of S. Enteritidis were present at each plant.

Sinorhizobium meliloti rpoE2 is necessary for H(2)O(2) stress resistance during the stationary growth phase.

RpoE2 is an extracytoplasmic sigma factor produced by Sinorhizobium meliloti during stationary growth phase. Its inactivation affected the synthesis of the superoxide dismutase, SodC, and catalase, KatC. The absence of SodC within the cell did not result in an increased sensitivity to extracellular superoxides. In contrast, the absence of KatC affected the resistance of S. meliloti to H(2)O(2) during the stationary growth phase. A katC strain behaved as an rpoE2 strain during an H(2)O(2) challenge, suggesting that the H(2)O(2) sensitivity of the rpoE2 strain resulted only from the lack of KatC in this strain.

Role of the Cj1371 periplasmic protein and the Cj0355c two-component regulator in the Campylobacter jejuni NCTC 11168 response to oxidative stress caused by paraquat.

Campylobacter jejuni is a microaerophilic pathogen representing one of the major causes of bacterial enteritis in humans. The oxidative stress response after exposure to paraquat, a strong oxidising agent, was analysed by two-dimensional protein electrophoresis and Maldi-ToF mass spectrometry. Oxidative stress and redox-related proteins were overexpressed: FldA flavodoxin and a pyruvate-flavodoxin oxidoreductase encoded by cj1476c. No increase in SodB expression was observed. An additional quantitative RT-PCR analysis showed an increase in katA but not in sodB expression. However, the sodB mutant was very sensitive to paraquat, its basal expression level being essential for oxidative stress resistance. Proteins related to iron homeostasis (Cft and a non-haem iron protein encoded by cj0012c) and general stress response (FusA and MreB) were found overexpressed. Interestingly, a two-component regulator encoded by cj0355c was differentially expressed in the presence of paraquat and could play a role in induction of the C. jejuni oxidative stress response. Virulence factors (CadF, FlaA and a VacJ homolog encoded by cj1371) were also found overexpressed under oxidative stress conditions and a cj1371 mutant showed increased sensitivity to paraquat, suggesting that the Cj1371 periplasmic protein could play a role in C. jejuni oxidative stress resistance.

The post-transcriptional regulator CsrA plays a central role in the adaptation of bacterial pathogens to different stages of infection in animal hosts.

The importance of Csr post-transcriptional systems is gradually emerging; these systems control a variety of virulence-linked physiological traits in many pathogenic bacteria. This review focuses on the central role that Csr systems play in the pathogenesis of certain bacteria and in the establishment of successful infections in animal hosts. Csr systems appear to control the 'switch' between different physiological states in the infection process; for example switching pathogens from a colonization state to a persistence state. Csr systems are controlled by two-component sensor/regulator systems and by non-coding RNAs. In addition, recent findings suggest that the RNA chaperone Hfq may play an integral role in Csr-mediated bacterial adaptation to the host environment.

Interrelations between glycine betaine catabolism and methionine biosynthesis in Sinorhizobium meliloti strain 102F34.

Methionine is produced by methylation of homocysteine. Sinorhizobium meliloti 102F34 possesses only one methionine synthase, which catalyzes the transfer of a methyl group from methyl tetrahydrofolate to homocysteine. This vitamin B(12)-dependent enzyme is encoded by the metH gene. Glycine betaine can also serve as an alternative methyl donor for homocysteine. This reaction is catalyzed by betaine-homocysteine methyl transferase (BHMT), an enzyme that has been characterized in humans and rats. An S. meliloti gene whose product is related to the human BHMT enzyme has been identified and named bmt. This enzyme is closely related to mammalian BHMTs but has no homology with previously described bacterial betaine methyl transferases. Glycine betaine inhibits the growth of an S. meliloti bmt mutant in low- and high-osmotic strength media, an effect that correlates with a decrease in the catabolism of glycine betaine. This inhibition was not observed with other betaines, like homobetaine, dimethylsulfoniopropionate, and trigonelline. The addition of methionine to the growth medium allowed a bmt mutant to recover growth despite the presence of glycine betaine. Methionine also stimulated glycine betaine catabolism in a bmt strain, suggesting the existence of another catabolic pathway. Inactivation of metH or bmt did not affect the nodulation efficiency of the mutants in the 102F34 strain background. Nevertheless, a metH strain was severely defective in competing with the wild-type strain in a coinoculation experiment.

Ecology of Listeria monocytogenes in the environment of raw poultry meat and raw pork meat processing plants.

The zoonotic Listeria monocytogenes is mainly transmitted to humans by the food-borne route. This bacterium was often found in the environment of food processing plants. Therefore the aims of this study were (i) the identification of environmental factors associated with L. monocytogenes contamination on working and non-working surfaces in poultry or pork processing plants and (ii) the understanding of its survival in such environments. The physicochemical risk profiles showed that a surface in resin or plastic, rather than uneven, with organic residues, with a neutral pH, a low temperature and a high hygrometry was associated with L. monocytogenes contamination.