Cellular Adaptations to Cytoplasmic Mg2+ Limitation

Mg2+ is the most abundant divalent cation in living cells. It is essential for charge neutralization, macromolecule stabilization, and the assembly and activity of ribosomes and as a cofactor for enzymatic reactions. When experiencing low cytoplasmic Mg2+, bacteria adopt two main strategies: They increase the abundance and activity of Mg2+ importers and decrease the abundance of Mg2+-chelating ATP and rRNA. These changes reduce regulated proteolysis by ATP-dependent proteases and protein synthesis in a systemic fashion. In many bacterial species, the transcriptional regulator PhoP controls expression of proteins mediating these changes. The 5′ leader region of some mRNAs responds to low cytoplasmic Mg2+ or to disruptions in translation of open reading frames in the leader regions by furthering expression of the associated coding regions, which specify proteins mediating survival when the cytoplasmic Mg2+ concentration is low. Microbial species often utilize similar adaptation strategies to cope with low cytoplasmic Mg2+ despite relying on different genes to do so.

The Serine Biosynthesis of Paenibacillus polymyxa WLY78 Is Regulated by the T-Box Riboswitch

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.

Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei

Casposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction. However, deletions of two-thirds of the target site were fully functional. Various single-stranded 6-FAM-labelled oligonucleotides derived from casposon terminal inverted repeats were as efficiently incorporated as duplexes into the target site. This result suggests that, as in the case of spacer insertion by the CRISPR Cas1–Cas2 integrase, casposon integration involves splaying of the casposon termini, with single-stranded ends being the actual substrates. The sequence critical for incorporation was limited to the five terminal residues derived from the 3′ end of the casposon. Furthermore, we characterize the casposase from Nitrosopumilus koreensis, a marine member of the phylum Thaumarchaeota, and show that it shares similar properties with the A. boonei enzyme, despite belonging to a different family. These findings further reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the CRISPR–Cas systems.

Conserved DNA motifs in the type II-A CRISPR leader region

The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3′ end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3′ leader end: ATTTGAG (noted previously in the CRISPR1 locus ofStreptococcus thermophilusDGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus ofS. thermophilusDGCC7710. A third group with a very short CG DNA conservation at the 3′ leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci.

Identification of a Residue (Glu60) in TRAP Required for Inducing Efficient Transcription Termination at the trp Attenuator Independent of Binding Tryptophan and RNA

ABSTRACT Transcription of the tryptophan (trp) operon in Bacillus subtilis is regulated by an attenuation mechanism. Attenuation is controlled by the t rp RNA-binding attenuation protein (TRAP). TRAP binds to a site in the 5′ leader region of the nascent trp transcript in response to the presence of excess intracellular tryptophan. This binding induces transcription termination upstream of the structural genes of the operon. In prior attenuation models, the role of TRAP was only to alter the secondary structure of the leader region RNA so as to promote formation of the trp attenuator, which was presumed to function as an intrinsic terminator. However, formation of the attenuator alone has been shown to be insufficient to induce efficient termination, indicating that TRAP plays an additional role in this process. To further examine the function of TRAP, we performed a genetic selection for mutant TRAPs that bind tryptophan and RNA but show diminished termination at the trp attenuator. Five such TRAP mutants were obtained. Four of these have substitutions at Glu60, three of which are Lys (E60K) substitutions and the fourth of which is a Val (E60V) substitution. The fifth mutant obtained contains a substitution at Ile63, which is on the same β-strand of TRAP as Glu60. Purified E60K TRAP binds tryptophan and RNA with properties similar to those of the wild type but is defective at inducing termination at the trp attenuator in vitro. IMPORTANCE Prior models for attenuation control of the B. subtilis trp operon suggested that the only role for TRAP is to bind to the leader region RNA and alter its folding to induce formation of an intrinsic terminator. However, several recent studies suggested that TRAP plays an additional role in the termination mechanism. We hypothesized that this function could involve residues in TRAP other than those required to bind tryptophan and RNA. Here we obtained TRAP mutants with alterations at Glu60 that are deficient at inducing termination in the leader region while maintaining tryptophan and RNA binding properties similar to those of the WT protein. These studies provide additional evidence that TRAP-mediated transcription termination at the trp attenuator is neither intrinsic nor Rho dependent.