Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism

Using an experimental approach, we investigated the RNome of the pathogen Staphylococcus aureus to identify 30 small RNAs (sRNAs) including 14 that are newly confirmed. Among the latter, 10 are encoded in intergenic regions, three are generated by premature transcription termination associated with riboswitch activities, and one is expressed from the complementary strand of a transposase gene. The expression of four sRNAs increases during the transition from exponential to stationary phase. We focused our study on RsaE, an sRNA that is highly conserved in the bacillales order and is deleterious when over-expressed. We show that RsaE interacts in vitro with the 5′ region of opp3A mRNA, encoding an ABC transporter component, to prevent formation of the ribosomal initiation complex. A previous report showed that RsaE targets opp3B which is co-transcribed with opp3A. Thus, our results identify an unusual case of riboregulation where the same sRNA controls an operon mRNA by targeting two of its cistrons. A combination of biocomputational and transcriptional analyses revealed a remarkably coordinated RsaE-dependent downregulation of numerous metabolic enzymes involved in the citrate cycle and the folate-dependent one-carbon metabolism. As we observed that RsaE accumulates transiently in late exponential growth, we propose that RsaE functions to ensure a coordinate downregulation of the central metabolism when carbon sources become scarce

Nickel import in bacteria, structural study of extracytoplasmic nickel-binding proteins

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Dual Role of the Oligopeptide Permease Opp3 during Growth of Staphylococcus aureus in Milk▿

Staphylococcus aureus RN6390 presents a diauxic growth in milk, due to amino acid limitation. Inactivation of the oligopeptide permease Opp3 (dedicated to the nitrogen nutrition of the strain) not only affects the growth of the strain but also results in reduced expression levels of three major extracellular proteases

Promiscuous Nickel Import in Human Pathogens: Structure, Thermodynamics, and Evolution of Extracytoplasmic Nickel-Binding Proteins

International audienceIn human pathogenic bacteria, nickel is required for the activation of two enzymes, urease and [NiFe]-hydrogenase, necessary for host infection. Acquisition of Ni(II) is mediated by either permeases or ABC-importers, the latter including a subclass that involves an extracytoplasmic nickel-binding protein, Ni-BP. This study reports on the structure of three Ni-BPs from a diversity of human pathogens and on the existence of three new nickel-binding motifs. These are different from that previously described for Escherichia coli Ni-BP NikA, known to bind nickel via a nickelophore, and indicate a variegated ligand selectivity for Ni-BPs. The structures are consistent with ligand affinities measured in solution by calorimetry and challenge the hypothesis of a general requirement of nickelophores for nickel uptake by canonical ABC importers. Phylogenetic analyses showed that Ni-BPs have different evolutionary origins and emerged independently from peptide-binding proteins, possibly explaining the promiscuous behavior of this class of Ni(II) carriers

Visualization of the role of host heme on the virulence of the heme auxotroph Streptococcus agalactiae

International audienceHeme is essential for several cellular key functions but is also toxic. Whereas most bacterial pathogens utilize heme as a metabolic cofactor and iron source, the impact of host heme during bacterial infection remains elusive. The opportunist pathogen Streptococcus agalactiae does not synthesize heme but still uses it to activate a respiration metabolism. Concomitantly, heme toxicity is mainly controlled by the HrtBA efflux transporter. Here we investigate how S. agalactiae manages heme toxicity versus benefits in the living host. Using bioluminescent bacteria and heme-responsive reporters for in vivo imaging, we show that the capacity of S. agalactiae to overcome heme toxicity is required for successful infection, particularly in blood-rich organs. Host heme is simultaneously required, as visualized by a generalized infection defect of a respiration-negative mutant. In S. agalactiae, HrtBA expression responds to an intracellular heme signal via activation of the two-component system HssRS. A hssRS promoter-driven intracellular luminescent heme sensor was designed to identify host compartments that supply S. agalactiae with heme. S. agalactiae acquires heme in heart, kidneys, and liver, but not in the brain. We conclude that S. agalactiae response to heme is organ-dependent, and its efflux may be particularly relevant in late stages of infection

Following Pathogen Development and Gene Expression in a Food Ecosystem: the Case of a Staphylococcus aureus Isolate in Cheese

Human intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on the in situ distribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate of Staphylococcus aureus, an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring of S. aureus populations and targeted gene expression. The combination of complementary techniques revealed that S. aureus localizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices

The Adc/Lmb System Mediates Zinc Acquisition in Streptococcus agalactiae and Contributes to Bacterial Growth and Survival

International audienceABSTRACT The Lmb protein of Streptococcus agalactiae is described as an adhesin that binds laminin, a component of the human extracellular matrix. In this study, we revealed a new role for this protein in zinc uptake. We also identified two Lmb homologs, AdcA and AdcAII, redundant binding proteins that combine with the AdcCB translocon to form a zinc-ABC transporter. Expression of this transporter is controlled by the zinc concentration in the medium through the zinc-dependent regulator AdcR. Triple deletion of lmb , adcA , and adcAII , or that of the adcCB genes, impaired growth and cell separation in a zinc-restricted environment. Moreover, we found that this Adc zinc-ABC transporter promotes S. agalactiae growth and survival in some human biological fluids, suggesting that it contributes to the infection process. These results indicated that zinc has biologically vital functions in S. agalactiae and that, under the conditions tested, the Adc/Lmb transporter constitutes the main zinc acquisition system of the bacterium. IMPORTANCE A zinc transporter, composed of three redundant binding proteins (Lmb, AdcA, and AdcAII), was characterized in Streptococcus agalactiae . This system was shown to be essential for bacterial growth and morphology in zinc-restricted environments, including human biological fluids

Pseudomonas aeruginosa zinc uptake in chelating environment is primarily mediated by the metallophore pseudopaline

Metal uptake is vital for all living organisms. In metal scarce conditions a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here we characterize a four-gene operon (PA4837-PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated with an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The pseudopaline system is involved in nickel uptake in poor media, and, most importantly, in zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as the main zinc importer under these metal scarce conditions

Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese▿

Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk. Staphylococcus aureus is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during S. aureus growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were ftsZ, pta, and gyrB, and these were used as internal controls for RT-qPCR of the genes sea and sed, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production