Small stable RNA maturation and turnover in Bacillus subtilis

Stable RNA maturation is a key process in the generation of functional RNAs, and failure to correctly process these RNAs can lead to their elimination through quality control mechanisms. Studies of the maturation pathways of ribosomal RNA and transfer RNA in Bacillus subtilis showed they were radically different from Escherichia coli and led to the identification of new B. subtilis‐specific enzymes. We noticed that, despite their important roles in translation, a number of B. subtilis small stable RNAs still did not have characterised maturation pathways, notably the tmRNA, involved in ribosome rescue, and the RNase P RNA, involved in tRNA maturation. Here, we show that tmRNA is matured by RNase P and RNase Z at its 5′ and 3′ extremities, respectively, whereas the RNase P RNA is matured on its 3′ side by RNase Y. Recent evidence that several RNases are not essential in B. subtilis prompted us to revisit maturation of the scRNA, a component of the signal recognition particle involved in co‐translational insertion of specific proteins into the membrane. We show that RNase Y is also involved in 3′ processing of scRNA. Lastly, we identified some of the enzymes involved in the turnover of these three stable RNAs.     

Selective cloning of genes encoding RNase H from Salmonella typhimurium, Saccharomyces cerevisiae and Escherichia coli rnh mutant

Summary We have cloned genes encoding RNase H from Escherichia coli rnh mutants, Salmonella typhimurium and Saccharomyces cerevisiae. Selection was accomplished by suppression of the temperature-sensitive growth phenotype of Escherichia coli strains containing the rnh-339::cat and either recB270 (Ts) or recC271 (Ts) mutations. RNases H from E. coli and S. typhimurium contained 155 amino acid residues and differed at only 11 positions. The S. cerevisiae and E. coli RNases H were about 30% homologous. A comparison of the amino acid sequences of several RNases H from cellular and retroviral sources revealed some strongly conserved regions as well as variable regions; the carboxyl-terminus was particularly variable. The overlapping, divergent promoter gene organization found in E. coli was observed to be present in S. typhimurium.     

Ribonucleases J1 and J2: two novel endoribonucleases in B.subtilis with functional homology to E.coli RNase E

Many prokaryotic organisms lack an equivalent of RNase E, which plays a key role in mRNA degradation in Escherichia coli. In this paper, we report the purification and identification by mass spectrometry in Bacillus subtilis of two paralogous endoribonucleases, here named RNases J1 and J2, which share functional homologies with RNase E but no sequence similarity. Both enzymes are able to cleave the B.subtilis thrS leader at a site that can also be cleaved by E.coli RNase E. We have previously shown that cleavage at this site increases the stability of the downstream messenger. Moreover, RNases J1/J2 are sensitive to the 5′ phosphorylation state of the substrate in a site-specific manner. Orthologues of RNases J1/J2, which belong to the metallo-β-lactamase family, are evolutionarily conserved in many prokaryotic organisms, representing a new family of endoribonucleases. RNases J1/J2 appear to be implicated in regulatory processing/maturation of specific mRNAs, such as the T-box family members thrS and thrZ, but may also contribute to global mRNA degradation.     

Design and Diversity in Bacterial Chemotaxis: A Comparative Study in Escherichia coli and Bacillus subtilis

Comparable processes in different species often involve homologous genes. One question is whether the network structure, in particular the feedback control structure, is also conserved. The bacterial chemotaxis pathways in E. coli and B. subtilis both regulate the same task, namely, excitation and adaptation to environmental signals. Both pathways employ many orthologous genes. Yet how these orthologs contribute to network function in each organism is different. To investigate this problem, we propose what is to our knowledge the first computational model for B. subtilis chemotaxis and compare it to previously published models for chemotaxis in E. coli. The models reveal that the core control strategy for signal processing is the same in both organisms, though in B. subtilis there are two additional feedback loops that provide an additional layer of regulation and robustness. Furthermore, the network structures are different despite the similarity of the proteins in each organism. These results demonstrate the limitations of pathway inferences based solely on homology and suggest that the control strategy is an evolutionarily conserved property.     

Design and diversity in bacterial chemotaxis: a comparative study in Escherichia coli and Bacillus subtilis

Comparable processes in different species often involve homologous genes. One question is whether the network structure, in particular the feedback control structure, is also conserved. The bacterial chemotaxis pathways in E. coli and B. subtilis both regulate the same task, namely, excitation and adaptation to environmental signals. Both pathways employ many orthologous genes. Yet how these orthologs contribute to network function in each organism is different. To investigate this problem, we propose what is to our knowledge the first computational model for B. subtilis chemotaxis and compare it to previously published models for chemotaxis in E. coli. The models reveal that the core control strategy for signal processing is the same in both organisms, though in B. subtilis there are two additional feedback loops that provide an additional layer of regulation and robustness. Furthermore, the network structures are different despite the similarity of the proteins in each organism. These results demonstrate the limitations of pathway inferences based solely on homology and suggest that the control strategy is an evolutionarily conserved property.     

Comparative studies of RNA polymerase subunits from various bacteria

Summary The molecular structure of RNA polymerases from Escherichia coli, Salmonella typhimurium, Salmonella anatum, Serratia marcescens, Aerobacter aerogenes, Proteus mirabilis and Bacillus subtilis were compared based on: i) inhibition of the enzyme activity by treatment with antibodies against E. coli RNA polymerase subunits; ii) analysis of antibody precipitates by sodium dodecyl sulfatepolyacrylamide gel electrophoresis; and iii) analysis of antibody precipitates by urea-isoelectrofocusing followed by sodium dodecyl sulfate-slab gel electrophoresis in the second dimension.All the bacterial RNA polymerases examined cross-react equally with anti-E. coli holopolymerase but exhibit different extents of cross-reaction with antibodies against individual subunits. Except for B. subtilis RNA polymerase, the molecular weight and isoelectric point of the enzyme subunits are close to those of E. coli polymerase. However, minor differences were found at least within the resolution of the techniques employed: S. anatum polymerase has subunit larger than E. coli subunit; P. mirabilis enzyme has subunit larger in size and more acidic in charge, and subunit smaller and more basic than corresponding E. coli subunits. The electrophoretic map of B. subtilis enzyme subunits is completely different from that of E. coli enzyme.     

Effect of nucleases on bacteria infected with bacteriophages

The effect of bacterial nucleases on bacteria infected by DNA- or RNA-containing bacteriophages with different serogroups was studied. Bacillary RNases have a strong inhibitory effect on RNA-containing bacteriophages. It was shown that nucleases suppressed the infection process of bacteria by bacteriophages M12, f2, PP7, and QB. The minimal inhibitory concentration ranged from 0.6 to 6 μg/mL. Bacterial ribonucleases have no impact on the development of DNA-containing bacteriophages PZ-A, PZ-B, P₃k, P118, and a lysogenic culture of Escherichia coli (λ) and Bacillus subtilis 168 (phi105). RNase from Bacillus pumilus did not inactivate bacteriophages Qβ and f2 in vitro and did not influence the adsorption on bacteriophages on the cell wall of the bacteria host E. coli AB301. The enzyme effect was shown at the level of bacteriophage infection of the host bacteria. Presumably, the phase between the adsorption and penetration of phage RNA into bacterial pili is the most sensitive to the effect of RNases.     

Differential antibacterial activity of genistein arising from global inhibition of DNA, RNA and protein synthesis in some bacterial strains

Antibacterial activities of various flavonoids have been reported previously, but mechanism(s) of their action on bacterial cells remain(s) largely unknown. Here, we investigated effects of genistein, an isoflavone, and representatives of other flavonoids: daidzein (another isoflavone), apigenin (a flavone), naringenin (a flavanone) and kaempferol (a flavonol), on commonly used laboratory strains of model bacterial species: Escherichia coli, Vibrio harveyi and Bacillus subtilis. We found that E. coli was resistant to all tested flavonoids at concentrations up to 0.1 mM, while high sensitivity of V. harveyi to most of them (except daidzein, which exhibited significantly less pronounced effect) was observed. Effects of the flavonoids on B. subtilis were relatively intermediate to the two extremes, i.e., E. coli and V. harveyi. Action of genistein on bacterial cells was investigated in more detail to indicate changed cell morphology (formation of filamentous cells) of V. harveyi and drastic inhibition of global synthesis of DNA and RNA as shortly as 15 min after addition of this isoflavone to a bacterial culture to a final concentration of 0.1 mM. Protein synthesis inhibition was also apparent, but delayed. Both cell morphology and synthesis of nucleic acids and proteins were unaffected in E. coli cultures under analogous conditions. Studies on cell survival suggest that genistein is a bacteriostatic agent rather than a bactericidal compound.     

Unraveling the potential and constraints associated with corn steep liquor as a nutrient source for industrial fermentations

Costly complex media components such as yeast extract and peptone are still widely used in industrial bioprocesses, despite their ill-defined composition. Side stream products such as corn steep liquor (CSL) present a compelling economical alternative that contains valuable nutrients required for microbial growth, that is, nitrogen and amino acids, but also vitamins, trace elements, and other minerals. However, as a side stream product, CSL may be subject to batch-to-batch variations and compositional heterogeneity. In this study, the Respiration Activity MOnitoring System designed for shake flasks (RAMOS) and 96-well microtiter plates (μTOM) were applied to investigate the potential and constraints of CSL utilization for two model microorganisms: E. coli and B. subtilis. Considering the dry substance content of complex nutrients involved, CSL-based media are more efficient in biomass production than the common lysogeny broth (LB) medium, containing 5 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl. At a glucose to CSL (glucose/CSL, g/g) ratio of 1/1 (g/g) and 2/1 (g/g), a secondary substrate limitation occurred in E. coli and B. subtilis cultivations, respectively. The study sheds light on differences in the metabolic activity of the two applied model organisms between varying CSL batches, which relate to CSL origin and production process, as well as the effect of targeted nutrient supplementation. Through a targeted nutrient supplementation, the most limiting component of the CSL-glucose medium used for these applied model microorganisms was identified to be ammonium nitrogen. This study proves the suitability of CSL as an alternative nutrient source for E. coli and B. subtilis. The RAMOS and μTOM technique detected differences between CSL batches, allowing easy and early identification of varying batches. A consistent performance of the CSL batches in E. coli and B. subtilis cultivations was demonstrated.     

Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo

Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.     

Impairment of ribosomal subunit synthesis in aminoglycoside-treated ribonuclease mutants of Escherichia coli

The bacterial ribosome is an important target for many antimicrobial agents. Aminoglycoside antibiotics bind to both 30S and 50S ribosomal subunits, inhibiting translation and subunit formation. During ribosomal subunit biogenesis, ribonucleases (RNases) play an important role in rRNA processing. E. coli cells deficient for specific processing RNases are predicted to have an increased sensitivity to neomycin and paromomycin. Four RNase mutant strains showed an increased growth sensitivity to both aminoglycoside antibiotics. E. coli strains deficient for the rRNA processing enzymes RNase III, RNase E, RNase G or RNase PH showed significantly reduced subunit amounts after antibiotic treatment. A substantial increase in a 16S RNA precursor molecule was observed as well. Ribosomal RNA turnover was stimulated, and an enhancement of 16S and 23S rRNA fragmentation was detected in E. coli cells deficient for these enzymes. This work indicates that bacterial RNases may be novel antimicrobial targets.     

Biosynthesis of secreted ribonucleases of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> and <Emphasis Type="Italic">Bacillus circulans</Emphasis> under nitrogen starvation

The level of biosynthesis of secreted guanyl-specific ribonucleases (RNases) of Bacillus intermedius (binases) and Bacillus circulans (RNases Bci) by recombinant B. subtilis strains increases under nitrogen starvation. The promoter of the binase gene carries the sequences homologous to the recognition sites of the regulatory protein TnrA, which regulates gene expression under growth limitation by nitrogen. Using the B. subtilis strain defective in protein TnrA, it has been shown that the regulatory protein TnrA is involved in the regulation of expression of the binase gene and the gene of RNase Bci. The TnrA regulation of expression of the RNase Bci gene is indirect, probably by means of the regulatory protein PucR. Thus, it has been established that at least two regulatory mechanisms activate the expression of the genes encoding the secreted RNases of spore-forming bacteria: a system of proteins homologous to the B. subtilis PhoP-PhoR, and regulation by a protein similar to the B. subtilis TnrA regulatory protein.     

Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

Single-stranded DNA-binding proteins (SSBs) are required for repair, recombination and replication in all organisms. Eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues. To our knowledge, phosphorylation of SSBs in bacteria has not been reported. A systematic search for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (ScSSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation of SSBs is a conserved process of post-translational modification in taxonomically distant bacteria.     

Mutations in domain V of the 23S ribosomal RNA of Bacillus subtilis that inactivate its protein folding property in vitro

The active site of a protein folding reaction is in domain V of the 23S rRNA in the bacterial ribosome and its homologs in other organisms. This domain has long been known as the peptidyl transferase center. Domain V of Bacillus subtilis is split into two segments, the more conserved large peptidyl transferase loop (RNA1) and the rest (RNA2). These two segments together act as a protein folding modulator as well as the complete domain V RNA. A number of site-directed mutations were introduced in RNA1 and RNA2 of B.subtilis, taking clues from reports of these sites being involved in various steps of protein synthesis. For example, sites like G2505, U2506, U2584 and U2585 in Escherichia coli RNA1 region are protected by deacylated tRNA at high Mg²⁺ concentration and A2602 is protected by amino acyl tRNA when the P site remains occupied already. Mutations A2058G and A2059G in the RNA1 region render the ribosome Ery^r in E.coli and Lnc^r in tobacco chloroplast. Sites in P loop G2252 and G2253 in E.coli are protected against modification by the CCA end of the P site bound tRNA. Mutations were introduced in corresponding nucleotides in B.subtilis RNA1 and RNA2 of domain V. The mutants were tested for refolding using unfolded protein binding assays with unfolded carbonic anhydrase. In the protein folding assay, the mutants showed partial to complete loss of this activity. In the filter binding assay for the RNA–refolding protein complex, the mutants showed an extent of protein binding that agreed well with their protein folding activity.     

Experiences with the on-line measurement of culture fluorescence during cultivation of Bacillus subtilis, Escherichia coli and Sporotrichum thermophile

Bacillus subtilis and Escherichia coli K12 (both transformed for human leukocyte interferon production) and Escherichia coli B/r and Sporotrichum thermophile (a deuteromycete) were cultivated in submersed culture and the culture fluorescence recorded on-line using a fluorometer. During the cultivation of B. subtilis the signal from the fluorometer correlated with cell density and interferon production and thus could be used for process control (interferon production). However, the culture fluorescence of the other organisms did not increase (S. thermophile), was too weak to be measured with the fluorometer used (E. coli transformed for interferon production), or the signal from the fluorometer was not an accurate measure of the culture fluorescence because of the accumulation of a fluorophor in the culture medium (E. coli B/r).     

Diversity in Chemotaxis Mechanisms among the Bacteria and Archaea

The study of chemotaxis describes the cellular processes that control the movement of organisms toward favorable environments. In bacteria and archaea, motility is controlled by a two-component system involving a histidine kinase that senses the environment and a response regulator, a very common type of signal transduction in prokaryotes. Most insights into the processes involved have come from studies of Escherichia coli over the last three decades. However, in the last 10 years, with the sequencing of many prokaryotic genomes, it has become clear that E. coli represents a streamlined example of bacterial chemotaxis. While general features of excitation remain conserved among bacteria and archaea, specific features, such as adaptational processes and hydrolysis of the intracellular signal CheY-P, are quite diverse. The Bacillus subtilis chemotaxis system is considerably more complex and appears to be similar to the one that existed when the bacteria and archaea separated during evolution, so that understanding this mechanism should provide insight into the variety of mechanisms used today by the broad sweep of chemotactic bacteria and archaea. However, processes even beyond those used in E. coli and B. subtilis have been discovered in other organisms. This review emphasizes those used by B. subtilis and these other organisms but also gives an account of the mechanism in E. coli.     

The Mode of Action of Pederin,a Drug Inhibiting Protein Synthesis in Eucaryotic Organisms

Abstract

Pederin, a toxic substance isolated from the insect Paederus fuscipes, inhibits growth of Saccharomyces cerevisiae and EUE cells but not of Bacillus subtilis. Protein synthesis in vitro appears to be inhibited by the drug in preparations obtained from organisms containing 80 S ribosomes (yeast, EUE cells and rat liver) but not in those from organisms endowed with 70 S ribosomes (E. coli and B. subtilis). Pederin inhibits protein synthesis at a stage subsequent to the formation of the ternary complex between ribosomes, aminoacyl-tRNA and messenger RNA. Resistance or susceptibility to the drug appears to be a characteristic of ribosomes.