A novel gyrase inhibitor from toxin-antitoxin system expressed by Staphylococcus aureus.

Toxin-antitoxin (TA) systems consist of a toxin inhibiting essential cellular functions (such as DNA, RNA and protein synthesis), and its cognate antitoxin neutralizing the toxicity. Recently, we identified a TA system termed TsbA/TsbT in the Staphylococcus aureus genome. The induction of the tsbT gene in Escherichia coli halted both DNA and RNA synthesis, reduced supercoiled plasmid and resulted in increasingly relaxed DNA. These results suggested that DNA gyrase was the target of TsbT. In addition, TsbT inhibited both E. coli and S. aureus DNA gyrase activity and induced linearization of plasmid DNA in vitro. Taken together, these results demonstrate that the TsbT toxin targets DNA gyrase in vivo. Site-directed mutagenesis experiments showed that the E27 and D37 residues in TsbT are critical for toxicity. Secondary structure prediction combining the analysis of vacuum-ultraviolet circular-dichroism spectroscopy and neural network method demonstrated that the 22nd-32nd residues of TsbT form an α-helix structure, and that the E27 residue is located around the centre of the α-helix segment. These findings give new insights not only into S. aureus TA systems, but also into bacterial toxins targeting DNA topoisomerases.

Assessment of prediction methods for protein structures determined by NMR in CASP14: Impact of AlphaFold2.

NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.

Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Evolution of the genetic code; Evidence from serine codon use disparity in Escherichia coli.

Among the 20 amino acids, three of them-leucine (Leu), arginine (Arg), and serine (Ser)-are encoded by six different codons. In comparison, all of the other 17 amino acids are encoded by either 4, 3, 2, or 1 codon. Peculiarly, Ser is separated into two disparate Ser codon boxes, differing by at least two-base substitutions, in contrast to Leu and Arg, of which codons are mutually exchangeable by a single-base substitution. We propose that these two different Ser codons independently emerged during evolution. In this hypothesis, at the time of the origin of life there were only seven primordial amino acids: Valine (coded by GUX [X = U, C, A or G]), alanine (coded by GCX), aspartic acid (coded by GAY [Y = U or C]), glutamic acid (coded by GAZ [Z = A or G]), glycine (coded by GGX), Ser (coded by AGY), and Arg (coded by CGX and AGZ). All of these were derived from GGX for glycine by single-base substitutions. Later in evolution, another class of Ser codons, UCX, were derived from alanine codons, GCX, distinctly different from the other primordial Ser codon, AGY. From the analysis of the Escherichia coli genome, we find extensive disparities in the usage of these two Ser codons, as some genes use only AGY for Ser in their genes. In contrast, others use only UCX, pointing to distinct differences in their origins, consistent with our hypothesis.

In-cell NMR as a sensitive tool to monitor physiological condition of Escherichia coli.

The in-cell NMR technique offers significant insights into the structure and function of heterologous proteins in the physiological intracellular environment at an atomic resolution. Escherichia coli (E. coli) is one of the most widely used host cells for heterologous protein expression in structural biological studies as well as for in-cell NMR studies to investigate fundamental structural characteristics and the physiochemistry of certain proteins and their intermolecular interactions under physiological conditions. However, in many cases, it is not easy to obtain well-resolved in-cell NMR spectra because the detectability and resolution of these spectra are significantly influenced by intracellular factors such as nonspecific intermolecular interactions. In this study, we re-examined the experimental parameters of E. coli in-cell NMR and found that the detectability and resolution of the NMR spectra clearly depended on the growth phase of the host cells. Furthermore, the detectability and resolution of the E. coli in-cell NMR spectra correlated with the soluble fraction amounts of the expressed target protein. These results indicate that the E. coli in-cell NMR spectrum of a target protein is a useful tool for monitoring the intracellular conditions of the host cell and for establishing the appropriate cultivation conditions for protein overexpression.

Mimicking cotranslational folding of prosubtilisin E in vitro.

Nascent polypeptides are synthesized on ribosomes starting at the N-terminus and simultaneously begin to fold during translation. We constructed N-terminal fragments of prosubtilisin E containing an intramolecular chaperone (IMC) at N-terminus to mimic cotranslational folding intermediates of prosubtilisin. The IMC-fragments of prosubtilisin exhibited progressive enhancement of their secondary structures and thermostabilities with increasing polypeptide length. However, even the largest IMC-fragment with 72 residues truncated from the C-terminus behaved as a molten globule, indicating the requirement of the C-terminal region to have a stable tertiary structure. Furthermore, truncation of the IMC in the IMC-fragments resulted in aggregation, suggesting that the IMC plays a crucial role to prevent misfolding and aggregation of cotranslational folding intermediates during translation of prosubtilisin polypeptide.

Protein structure prediction assisted with sparse NMR data in CASP13.

CASP13 has investigated the impact of sparse NMR data on the accuracy of protein structure prediction. NOESY and 15 N-1 H residual dipolar coupling data, typical of that obtained for 15 N,13 C-enriched, perdeuterated proteins up to about 40 kDa, were simulated for 11 CASP13 targets ranging in size from 80 to 326 residues. For several targets, two prediction groups generated models that are more accurate than those produced using baseline methods. Real NMR data collected for a de novo designed protein were also provided to predictors, including one data set in which only backbone resonance assignments were available. Some NMR-assisted prediction groups also did very well with these data. CASP13 also assessed whether incorporation of sparse NMR data improves the accuracy of protein structure prediction relative to nonassisted regular methods. In most cases, incorporation of sparse, noisy NMR data results in models with higher accuracy. The best NMR-assisted models were also compared with the best regular predictions of any CASP13 group for the same target. For six of 13 targets, the most accurate model provided by any NMR-assisted prediction group was more accurate than the most accurate model provided by any regular prediction group; however, for the remaining seven targets, one or more regular prediction method provided a more accurate model than even the best NMR-assisted model. These results suggest a novel approach for protein structure determination, in which advanced prediction methods are first used to generate structural models, and sparse NMR data is then used to validate and/or refine these models.

A CUGGU/UUGGU-specific MazF homologue from Methanohalobium evestigatum.

MazF is a sequence-specific endoribonuclease or mRNA interferase, which cleaves RNA at a specific sequence. Since the expression of a specific gene or a group of specific genes can be regulated by MazF, expanding the repertoire of recognition sequences by MazF mRNA interferases is highly desirable for biotechnological and medical applications. Here, we identified a gene for a MazF homologue (MazFme) from Methanohalobium evestigatum, an extremely halophilic archaeon. In order to suppress the toxicity of MazFme to the E. coli cells, the C-terminal half of the cognate antitoxin MazEme was fused to the N-terminal end of MazFme. Since the fusion of the C-terminal half of MazEme to MazFme was able to neutralize MazFme toxicity, the MazEme-MazFme fusion protein was expressed in a large amount without any toxic effects. After purification of the MazEme, the free MazFme RNA cleavage specificity was determined by primer extension and synthetic ribonucleotides, revealing that MazFme is a CUGGU/UUGGU-specific endoribonuclease.

Genome-Wide Screening for Identification of Novel Toxin-Antitoxin Systems in Staphylococcus aureus.

Toxin-antitoxin (TA) systems consist of toxin-inhibiting diverse cellular functions (e.g., DNA replication, transcription, and translation) and a noncoding RNA or protein antitoxin. TA systems are associated with various cellular events, such as stress responses, programmed cell death, and bacterial pathogenicity. Recent advances in genome sequencing and bioinformatics research have demonstrated that most bacteria harbor various kinds of TA modules on their chromosomes; however, there is little understanding of chromosomally encoded TA systems in the Gram-positive pathogen Staphylococcus aureus Here, we report on newly discovered S. aureus TA systems, each of which is composed of two proteins. Manual search and gene operon prediction analysis identified eight 2-gene operons as potential candidates for TA systems. Subsequently, using an Escherichia coli host killing and rescue assay, we demonstrated that four of the eight candidates worked as TA systems, designated tsaAT, tsbAT, tscAT, and tsdAT Moreover, the TsaT, TsbT, TscT, and TsdT toxins inhibited S. aureus growth, and the toxicity of TsbT was neutralized by coexpressing the tsbA gene in the native host, S. aureus Further, the bioinformatics analysis of the gene clusters revealed that TsaAT, TsbAT, TscAT, and TsdAT did not exhibit sequence similarity to known bacterial TA systems, and their homologues were present only within Staphylococcus species and not among any other bacteria. Our results further advance not only the understanding of S. aureus TA systems but also the study of unannotated TA systems in various bacterial species.IMPORTANCE Recent advances in genome sequencing and bioinformatics research have demonstrated that most pathogenic bacteria harbor a large number of chromosomally encoded toxin-antitoxin (TA) modules. However, little is known about the TA systems in S. aureus Here, we newly identified four S. aureus TA systems using a combination of manual base-by-base screening and functional analysis in E. coli Moreover, all toxins of the identified TA systems caused growth inhibition in the native host S. aureus Although the newly identified TA systems did not exhibit sequence similarity with known bacterial TA systems, their orthologues were conserved only among other Staphylococcus species, indicating their uniqueness to staphylococci. Our approach opens the possibility for studying unannotated TA systems in various bacterial species.

Combining Evolutionary Covariance and NMR Data for Protein Structure Determination.

Accurate protein structure determination by solution-state NMR is challenging for proteins greater than about 20kDa, for which extensive perdeuteration is generally required, providing experimental data that are incomplete (sparse) and ambiguous. However, the massive increase in evolutionary sequence information coupled with advances in methods for sequence covariance analysis can provide reliable residue-residue contact information for a protein from sequence data alone. These "evolutionary couplings (ECs)" can be combined with sparse NMR data to determine accurate 3D protein structures. This hybrid "EC-NMR" method has been developed using NMR data for several soluble proteins and validated by comparison with corresponding reference structures determined by X-ray crystallography and/or conventional NMR methods. For small proteins, only backbone resonance assignments are utilized, while for larger proteins both backbone and some sidechain methyl resonance assignments are generally required. ECs can be combined with sparse NMR data obtained on deuterated, selectively protonated protein samples to provide structures that are more accurate and complete than those obtained using such sparse NMR data alone. EC-NMR also has significant potential for analysis of protein structures from solid-state NMR data and for studies of integral membrane proteins. The requirement that ECs are consistent with NMR data recorded on a specific member of a protein family, under specific conditions, also allows identification of ECs that reflect alternative allosteric or excited states of the protein structure.

Identification of MazF Homologue in Legionella pneumophila Which Cleaves RNA at the AACU Sequence.

MazF is a sequence-specific endoribonuclease that is widely conserved in bacteria and archaea. Here, we found an MazF homologue (MazF-lp; LPO-p0114) in Legionella pneumophila. The mazF-lp gene overlaps 14 base pairs with the upstream gene mazE-lp (MazE-lp; LPO-p0115). The induction of mazF-lp caused cell growth arrest, while mazE-lp co-induction recovered cell growth in Escherichia coli. In vivo and in vitro primer extension experiments showed that MazF-lp is a sequence-specific endoribonuclease cleaving RNA at AACU. The endoribonuclease activity of purified MazF-lp was inhibited by purified MazE-lp. We found that MazE-lp and the MazEF-lp complex specifically bind to the palindromic sequence present in the 5'-untranslated region of the mazEF-lp operon. MazE-lp and MazEF-lp both likely function as a repressor for the mazEF-lp operon and for other genes, including icmR, whose gene product functions as a secretion chaperone for the IcmQ pore-forming protein, by specifically binding to the palindromic sequence in 5'-UTR of these genes.

Translation-dependent mRNA cleavage by YhaV in Escherichia coli.

Many bacteria have toxin-antitoxin (TA) systems, where toxin gene expression inhibits their own cell growth. mRNA is one of the well-known targets of the toxins in the type II toxin-antitoxin systems. Here, we examined the ribosome dependency of the endoribonuclease activity of YhaV, one of the toxins in type II TA systems, on mRNA in vitro and in vivo. A polysome profiling assay revealed that YhaV is bound to the 70S ribosomes and 50S ribosomal subunits. Moreover, we found that while YhaV cleaves ompF and lpp mRNAs in a translation-dependent manner, they did not cleave the 5' untranslated region in primer extension experiments. From these results, we conclude that YhaV is a ribosome-dependent toxin that cleaves mRNA in a translation-dependent manner.

Toxins of Prokaryotic Toxin-Antitoxin Systems with Sequence-Specific Endoribonuclease Activity.

Protein translation is the most common target of toxin-antitoxin system (TA) toxins. Sequence-specific endoribonucleases digest RNA in a sequence-specific manner, thereby blocking translation. While past studies mainly focused on the digestion of mRNA, recent analysis revealed that toxins can also digest tRNA, rRNA and tmRNA. Purified toxins can digest single-stranded portions of RNA containing recognition sequences in the absence of ribosome in vitro. However, increasing evidence suggests that in vivo digestion may occur in association with ribosomes. Despite the prevalence of recognition sequences in many mRNA, preferential digestion seems to occur at specific positions within mRNA and also in certain reading frames. In this review, a variety of tools utilized to study the nuclease activities of toxins over the past 15 years will be reviewed. A recent adaptation of an RNA-seq-based technique to analyze entire sets of cellular RNA will be introduced with an emphasis on its strength in identifying novel targets and redefining recognition sequences. The differences in biochemical properties and postulated physiological roles will also be discussed.

Characterization of YjjJ toxin of Escherichia coli.

Reminiscent of eukaryotic apoptotic programmed cell death, bacteria also contain a large number of suicide genes, which are in general co-expressed with their cognate antitoxin genes. These systems called the toxin-antitoxin (TA) systems are associated with cellular dormancy, and play major roles in biofilm formation and persistent multidrug resistance of many human pathogens. In recent years, the study on TA system toxins has become a hot topic due to the health implications of these toxins by virtue of their role in bacterial pathogenicity. Here we report functional characterization of a hitherto uncharacterized Escherichia coli TA toxin, YjjJ. YjjJ exhibits several uncommon properties: (i) unlike the genes encoding most type II TA system toxins, the gene encoding YjjJ is present as a single gene and not in an operon, (ii) despite being a homolog of the well-characterized toxin HipA, YjjJ seems to have different cellular target(s), and (iii) HipB, the cognate antitoxin of HipA, also acts as an antitoxin for YjjJ. This forms a basis for an interesting next step in the study of TA systems with respect to cross-regulation between various TA systems and the evolutionary as well as clinical significance of these observations.

Deletion of mazF increases Staphylococcus aureus biofilm formation in an ica-dependent manner.

Toxin-antitoxin (TA) systems are composed of a toxin that inhibits an essential cellular process (e.g. DNA replication, transcription, membrane integrity) and its cognate antitoxin that neutralizes the effect of the toxin. Staphylococcus aureus harbors two types of chromosomally encoded TA systems, namely mazEFsa encoding a UACAU-specific mRNA interferase and two paralogous genes of yefM-yoeBsa encoding a ribosome-dependent endoribonuclease system. However, little is known about the physiological role of MazEFsa and YefM-YoeBsa in S. aureus. Upon characterizing the phenotypes of single, double and triple gene deletion mutants, we found that mazFsa deletion led to increased biofilm formation. Subsequently, transcriptional analysis revealed that expression of intercellular adhesin (ica) gene, icaADBC, increased in a mazFsa deletion mutant. mazFsa/icaADBC double gene deletion and genetic complementation approaches provided convincing evidence that increased biofilm formation was caused by an increase in polysaccharide intercellular adhesin synthesized by icaADBC-encoded proteins. Furthermore, through the use of alanine substitutions at the conserved active residues of MazFsa, our results suggested that ica-mediated biofilm formation depended on the mRNA interferase activity of MazFsa. These findings give new insights not only into the physiological role of MazEFsa in S. aureus, but also into the regulatory mechanism of ica-dependent biofilm formation.

Designing of a single gene encoding four functional proteins.

In the genomes of some organisms such as bacteriophages and bacteria, a DNA sequence is able to encode two different proteins, indicating that genetic information is compacted in DNA twice denser than in usual DNA. In theory, a DNA sequence has a maximal capacity to produce six different mRNAs, however, it is an intriguing question how many of these mRNAs are able to synthesize functional proteins. Here, we design a DNA sequence encoding four collagen-like proteins, two, (Gly-Arg-Pro)n and (Gly-Ala-Pro)n, from a sense mRNA and the other two, also (Gly-Arg-Pro)n and (Gly-Ala-Pro)n from its antisense mRNA, all of which are expected to form triple-helical structures unique to collagens. Other designs such as the combination of (Gly-Arg-Pro)n, (Gly-Val-Pro)n, (Gly-Thr-Pro)n and (Gly-Arg-Pro)n are also possible. The proposed DNA sequence is considered to contain the most compact genetic information ever created.

The role of the loop 1 region in MazFbs mRNA interferase from Bacillus subtilis in recognition of the 3' end of the RNA substrate.

MazFbs is an mRNA interferase from Bacillus subtilis specifically recognizing UACAU. The X-ray structure of its complex with an RNA substrate has been also solved. When its amino acid sequence is compared with that of MazFhw, an mRNA interferase from a highly halophilic archaeon, recognizing UUACUCA, the 9-residue loop-1 region is highly homologous except that the V16V17 sequence in MazFbs is replaced with TK in MazFhw. Thus, we examined the role of the VV sequence in RNA substrate recognition by replacing it with TK, GG, AA or LL. The substitution mutants thus constructed showed significant differences in cleavage specificity using MS2 phage RNA. The primer extension analysis of the cleavage sites revealed that the VV sequence plays an important role in the recognition of the 3'-end base of the RNA substrate.

The first discovery of RNA interference by RNA restriction enzymes to inhibit protein synthesis.

In this article, I review how an RNA restriction enzyme, a highly sequence-specific endoribonuclease, was for the first time discovered in 2003 and how the concept of RNA interference using RNA restriction enzymes or mRNA interferases has been developed.

The first demonstration of the existence of reverse transcriptases in bacteria.

It has been long thought that reverse transcriptases are unique to the eukaryotes. However, through our research on a peculiar single stranded DNA called msDNA in Myxococcus xanthus, it was predicted that its synthesis requires reverse transcriptases. Subsequently, Lim and Maas as well as our group demonstrated the existence of reverse transcriptases for the production of msDNA. In this review, I describe how the discovery of msDNA led to the discovery of reverse transcriptases in bacteria and discuss the evolutionary significance of the discovery of revise transcriptases in bacteria.

Preferential use of minor codons in the translation initiation region of human genes.

More than 31,000 protein-coding sequences (CCDS) have been identified in the human genome. Here, we analyzed codon usage in all human CCDS and found that there is a preferential usage of minor codons for Ala (CGC), Pro (CCG), Ser (UCG), and Thr (ACG) in the initial 50-codon sequences of the CCDS. These codons, with consensus XCG sequences, are most infrequently used among their synonymous codons. Thus, the tRNA concentrations per codon are considered to be highest for the minor codons for Ala, Pro, Ser and Thr in comparison with other synonymous codons for each of them to enhance the translation efficiency. This suggests that human genes are regulated at the level of translation by preferentially using minor codons within the first 50 codons of the CCDS. This hypothesis was experimentally confirmed by comparing the expression of the luciferase gene encoded by minor codons with that encoded by major codons.