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.

Resistance to organic hydroperoxides requires ohr and ohrR genes in Sinorhizobium meliloti.

BACKGROUND: Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Medicago sativa. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H2O2 and superoxides has been extensively studied in S. meliloti. In contrast resistance to organic peroxides has not been investigated while S. meliloti genome encodes putative organic peroxidases. Organic peroxides are produced by plants and are highly toxic. The resistance to these oxygen radicals has been studied in various bacteria but never in plant nodulating bacteria. RESULTS: In this study we report the characterisation of organic hydroperoxide resistance gene ohr and its regulator ohrR in S. meliloti. The inactivation of ohr affects resistance to cumene and ter-butyl hydroperoxides but not to hydrogen peroxide or menadione in vitro. The expression of ohr and ohrR genes is specifically induced by organic peroxides. OhrR binds to the intergenic region between the divergent genes ohr and ohrR. Two binding sites were characterised. Binding to the operator is prevented by OhrR oxidation that promotes OhrR dimerisation. The inactivation of ohr did not affect symbiosis and nitrogen fixation, suggesting that redundant enzymatic activity exists in this strain. Both ohr and ohrR are expressed in nodules suggesting that they play a role during nitrogen fixation. CONCLUSIONS: This report demonstrates the significant role Ohr and OhrR proteins play in bacterial stress resistance against organic peroxides in S. meliloti. The ohr and ohrR genes are expressed in nodule-inhabiting bacteroids suggesting a role during nodulation.

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.

Proteomic alterations explain phenotypic changes in Sinorhizobium meliloti lacking the RNA chaperone Hfq.

The ubiquitous bacterial RNA-binding protein Hfq is involved in stress resistance and pathogenicity. In Sinorhizobium meliloti, Hfq is essential for the establishment of symbiosis with Medicago sativa and for nitrogen fixation. A proteomic analysis identifies 55 proteins with significantly affected expression in the hfq mutant; most of them are involved in cell metabolism or stress resistance. Important determinants of oxidative stress resistance, such as CysK, Gsh, Bfr, SodC, KatB, KatC, and a putative peroxiredoxine (SMc00072), are downregulated in the hfq mutant. The hfq mutant is affected for H(2)O(2), menadione, and heat stress resistance. Part of these defects could result from the reductions of rpoE1, rpoE2, rpoE3, and rpoE4 expression levels in the hfq mutant. Some proteins required for efficient symbiosis are reduced in the hfq mutant, contributing to the drastic defect in nodulation observed in this mutant.

The Sinorhizobium meliloti RNA chaperone Hfq mediates symbiosis of S. meliloti and alfalfa.

There exist commonalities between symbiotic Sinorhizobium meliloti and pathogenic Brucella bacteria in terms of extensive gene synteny and the requirements for intracellular survival in their respective hosts. The RNA chaperone Hfq is essential for virulence for several bacterial groups, including Brucella; however, its role in S. meliloti has not been investigated. Our studies of an S. meliloti loss-of-function hfq mutant have revealed that Hfq plays a key role in the establishment of the symbiosis between S. meliloti and its host Medicago sativa. S. meliloti Hfq is involved in controlling the population density under a free-living state and affects the growth parameters and nodulation. An hfq mutant poorly colonizes the infection threads that are necessary for the bacteria to invade the developing nodule. An hfq mutant is severely impaired in its ability to invade plant cells within the nodule, which leads to the formation of small, ineffective nodules unable to fix nitrogen. In culture, the hfq mutant did not accumulate transcripts of nifA, which encodes a key regulator necessary for nitrogen fixation. Hfq may be involved in regulation of several proteins relevant to hfq mutant phenotypes. The crucial role of Hfq in symbiosis suggests that small regulatory RNAs are important for its interactions with its plant host.

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.

Characterization and expression patterns of Sinorhizobium meliloti tmRNA (ssrA).

tmRNA (ssrA) in Sinorhizobium meliloti is a small RNA annotated by homology with the Bradyrhizobium japonicum sra molecule. Here, this molecule is described in Sinorhizobium meliloti as a model for such molecules in Alphaproteobacteria subgroup-2. Northern blot analysis and mapping of both 5' and 3' ends of this tmRNA allow the identification of two pieces: a 214 nt mRNA-like domain and an 82 nt tRNA-like domain, both highly stable, whereas the premature form is unstable. Transcriptional studies reveal that Sinorhizobium meliloti tmRNA is mainly expressed during growth resumption, replication initiation and various stress responses.

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.

Glucose 6-phosphate dehydrogenase is required for sucrose and trehalose to be efficient osmoprotectants in Sinorhizobium meliloti.

Inactivation of the zwf gene in Sinorhizobium meliloti induces an osmosensitive phenotype and the loss of osmoprotection by trehalose and sucrose, but not by ectoine and glycine betaine. This phenotype is not linked to a defect in the biosynthesis of endogenous solutes. zwf expression is induced by high osmolarity, sucrose and trehalose, but is repressed by betaine. A zwf mutant is more sensitive than its parental strain to superoxide ions, suggesting that glucose 6-phosphate dehydrogenase involvement in the osmotic response most likely results from the production of reactive oxygen species during osmotic stress.

Site-specific integration of corynephage phi16: construction of an integration vector.

Phi16, a temperate phage induced from Corynebacterium glutamicum ATCC 21792, lysogenizes its host via site-specific recombination. The phage attachment site, attP, was located to a 6.5 kb BamHI fragment of the phi16 genome. This fragment also contained phi16 integrative functions. The minimal phage DNA fragment required for integration was defined. This 1630 bp region contained a large open reading frame, int, encoding a protein of 416 amino acids with similarity in its carboxyl-terminal domain to tyrosine recombinases and particularly to the Xer recombinases. The comparison of the nucleotide sequences of attB, attL, attR, and attP identified a common 29 bp sequence, the core sequence. It lies 11 bp downstream of the 3' end of the integrase gene. phi16 integrase was shown to catalyse site-specific integration in trans to attP with an efficiency of 5x10(3) integrants per microg DNA. The integrating fragment catalysed integration in several Corynebacterium strains that are not infected by phi16, thus enlarging the host spectrum of integrating vectors derived from phi16. In these strains, the phi16 attB site was located in a conserved intergenic region and lies downstream of a clp gene.

pBLA8, from Brevibacterium linens, belongs to a gram-positive subfamily of ColE2-related plasmids.

A 3.1 kb DNA fragment from pBLA8, a Brevibacterium linens cryptic plasmid, containing all the information required for autonomous replication was cloned and sequenced. Using deletion analysis, the fragment essential and sufficient for autonomous replication was delimited to 1.5 kb. This fragment is characterized by the presence of an ori site located upstream of an operon encoding two proteins, RepA and RepB, both essential for replication. Based on structural similarities and a strong conservation of ori, RepA and RepB, pBLA8 was assigned to a new subfamily of the ColE2 plasmid family. This subfamily is distinguished by the requirement for two Rep proteins and the location of an ori site upstream of the repAB operon. RepA is thought to encode primase activity, whereas RepB could be a DNA-binding protein. An Escherichia coli-B. linens shuttle vector, derived from pBLA8, was constructed. Its host spectrum was extended to Arthrobacter species.