MqsR, a Crucial Regulator for Quorum Sensing and Biofilm Formation, Is a GCU-specific mRNA Interferase in Escherichia coli

The mqsR gene has been shown to be positively regulated by the quorum-sensing autoinducer AI-2, which in turn activates a two-component system, the qseB-qseC operon. This operon plays an important role in biofilm formation in Escherichia coli. However, its cellular function has remained unknown. Here, we found that 1 base downstream of mqsR there is a gene, ygiT, that is co-transcribed with mqsR. Induction of mqsR caused cell growth arrest, whereas ygiT co-induction recovered cell growth. We demonstrate that MqsR (98 amino acid residues), which has no homology to the well characterized mRNA interferase MazF, is a potent inhibitor of protein synthesis that functions by degrading cellular mRNAs. In vivo and in vitro primer extension experiments showed that MqsR is an mRNA interferase specifically cleaving mRNAs at GCU. The mRNA interferase activity of purified MqsR was inhibited by purified YgiT (131 residues). MqsR forms a stable 2:1 complex with YgiT, and the complex likely functions as a repressor for the mqsR-ygiT operon by specifically binding to two different palindromic sequences present in the 5′-untranslated region of this operon.It has been reported that quorum sensing is involved in biofilm formation (1–4). mqsR expression was found to be induced by 8-fold in biofilms (5) and also by the quorum-sensing signal autoinducer AI-2, which is a species-nonspecific signaling molecule produced by both Gram-negative and Gram-positive bacteria, including Escherichia coli (6). It has been reported that induction of mqsR activates a two-component system, the qseB-qseC operon, which is known to play an important role in biofilm formation (6). Thus, it has been proposed that MqsR (98 amino acid residues) is a regulator of biofilm formation because it activates qseB, which controls the flhDC expression required for motility and biofilm formation in E. coli (6). However, the cellular function of MqsR has remained unknown.Interestingly, all free-living bacteria examined to date contain a number of suicide or toxin genes in their genomes (7, 8). Many of these toxins are co-transcribed with their cognate antitoxins in an operon (termed toxin-antitoxin (TA)² operon) and form a stable complex in the cell, so their toxicity is subdued under normal growth conditions (9–11). However, the stability of antitoxins is substantially lower than that of their cognate toxins, so any stress causing cellular damage or growth inhibition that induces proteases alters the balance between toxin and antitoxin, leading to toxin release in the cell.To date, 16 (12) TA systems have been reported on the E. coli genome, including relB-relE (13, 14), chpBI-chpBK (15), mazEF (16–18), yefM-yoeB (19, 20), dinJ-yafQ (21, 22), hipBA and hicAB (23, 24), prlF-yhaV (25), and ybaJ-hha (26). Interestingly, all of these TA operons appear to use similar modes of regulation: the formation of complexes between antitoxins and their cognate toxins to neutralize toxin activity and the ability of TA complexes to autoregulate their expression. The cellular targets of some toxins have been identified. CcdB directly interacts with gyrase A and blocks DNA replication (27, 28). RelE, which by itself has no endoribonuclease activity, appears to act as a ribosome-associating factor that promotes mRNA cleavage at the ribosome A-site (13, 29, 30). PemK (31), ChpBK (15), and MazF (32) are unique among toxins because they target cellular mRNAs for degradation by functioning as sequence-specific endoribonucleases to effectively inhibit protein synthesis and thereby cell growth.MazF, ChpBK, and PemK have been characterized as sequence-specific endoribonucleases that cleave mRNA at the ACA, ACY (Y is U, A, or G), and UAH (H is C, A, or U) sequences, respectively. They are completely different from other known endoribonucleases such as RNases E, A, and T1, as these toxins function as protein synthesis inhibitors by interfering with the function of cellular mRNAs. It is well known that small RNAs, such as mRNA-interfering cRNA (33), microRNA (34), and small interfering RNA (35), interfere with the function of specific RNAs. These small RNAs bind to specific mRNAs to inhibit their expression. Ribozymes also act on their target RNAs specifically and interfere with their function (36). Therefore, MazF, ChpBK, and PemK homologs form a novel endoribonuclease family that exhibits a new mRNA-interfering mechanism by cleaving mRNAs at specific sequences. Thus, they have been termed “mRNA interferases” (2).During our search for TA systems on the E. coli genome, we found that the mqsR gene is co-transcribed with a downstream gene, ygiT. These two genes appear to function as a TA system, as their size is small (98 residues for MqsR and 131 residues for YgiT) and their respective open reading frames are separated by 1 bp. In this study, we demonstrate that MqsR-YgiT is a new E. coli TA system consisting of a toxin, MqsR, and an antitoxin, YgiT. Moreover, we identify MqsR as a novel mRNA interferase that does not exhibit homology to MazF. This toxin cleaves RNA at GCU sequences in vivo and in vitro. The implication of this finding as to how this mRNA interferase is involved in cell physiology and biofilm formation will be discussed.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Dominant-negative Inhibitors of the Clostridium perfringens ?-Toxin

The Clostridium perfringens ϵ-toxin is responsible for a severe, often lethal intoxication. In this study, we characterized dominant-negative inhibitors of the ϵ-toxin. Site-specific mutations were introduced into the gene encoding ϵ-toxin, and recombinant proteins were expressed in Escherichia coli. Paired cysteine substitutions were introduced at locations predicted to form a disulfide bond. One cysteine in each mutant was introduced into the membrane insertion domain of the toxin; the second cysteine was introduced into the protein backbone. Mutant proteins with cysteine substitutions at amino acid positions I51/A114 and at V56/F118 lacked detectable cytotoxic activity in a MDCK cell assay. Cytotoxic activity could be reconstituted in both mutant proteins by incubation with dithiothreitol, indicating that the lack of cytotoxic activity was attributable to the formation of a disulfide bond. Fluorescent labeling of the cysteines also indicated that the introduced cysteines participated in a disulfide bond. When equimolar mixtures of wild-type ϵ-toxin and mutant proteins were added to MDCK cells, the I51C/A114C and V56C/F118C mutant proteins each inhibited the activity of wild-type ϵ-toxin. Further analysis of the inhibitory activity of the I51C/A114C and V56C/F118C mutant proteins indicated that these proteins inhibit the ability of the active toxin to form stable oligomeric complexes in the context of MDCK cells. These results provide further insight into the properties of dominant-negative inhibitors of oligomeric pore-forming toxins and provide the basis for developing new therapeutics for treating intoxication by ϵ-toxin.The Clostridium perfringens ϵ-toxin is one of the most potent bacterial toxins (1, 2). The ϵ-toxin can lead to a fatal enterotoxemia characterized by widespread vascular permeability and edema in the heart, lungs, brain, and kidneys (3–6). The disease most frequently affects livestock animals, though the toxin may also affect humans (7–9). Because of its extreme potency and the possibility of intoxicating humans, the C. perfringens ϵ-toxin is considered a select agent by the United States Department of Health and Human Services. A vaccine currently is approved for veterinary use, though multiple immunizations are required to provide long-term immunity (10–13). There also is an antitoxin approved for veterinary use. However, in the event that an animal exhibits symptoms of intoxication by ϵ-toxin, it is typically too late for the current antitoxin to be effective, and use of the antitoxin is typically limited to prophylactic treatment of unvaccinated animals within a herd (14). There is no treatment currently approved for use in humans. Thus, alternative countermeasures are needed that inhibit the activity of the toxin.One alternative method of countering the cytotoxic activity of bacterial toxins is through dominant-negative inhibitors. Dominant-negative inhibitors are non-cytotoxic mutant forms of active toxins that are able to inhibit the activity of wild-type toxin when the two proteins are mixed together. Such dominant-negative inhibitors have been described for a diverse set of toxins, including Helicobacter pylori VacA (15–19), Bacillus anthracis anthrax toxin protective antigen (20–25), Bacillus thuringiensis Cry1Ab (26), and Escherichia coli ClyA cytotoxin (27). Like VacA, protective antigen, Cry1Ab, and ClyA, the ϵ-toxin assembles into oligomeric complexes containing multiple toxin monomers (28–30). In the case of VacA and protective antigen, the most extensively studied examples of toxins inhibited by dominant-negative mutants, the number of mutations that inactivate the toxins is substantially greater than the number of mutations that lead to a dominant-negative phenotype (16, 17, 24, 31, 32). Although many of the mutations leading to dominant-negative toxins are located within regions of the toxins that are believed to form the membrane insertion domain, some mutations that inactivate the toxins (but are not dominant-negative) also map within the predicted membrane insertion domains (24, 32). Thus, a deeper understanding of the nature of the dominant-negative phenotype is needed.In this study, we sought to generate dominant-negative mutants of the ϵ-toxin. We hypothesized that mutations within the membrane insertion domain of ϵ-toxin, particularly mutations that are expected to restrict movement of this domain, would lead to dominant-negative inhibitors. We expressed wild-type and site-specific mutants of the ϵ-toxin as recombinant proteins in E. coli. The recombinant proteins were purified, and cytotoxicity was assessed using an established cell culture assay. Using this approach, we identified mutant proteins that inhibited the activity of wild-type ϵ-toxin in vitro and determined the mechanism of inhibition.     

Geminin Stabilizes Cdt1 during Meiosis in Xenopus Oocytes

During the mitotic cell cycle, Geminin can act both as a promoter and inhibitor of initiation of DNA replication. As a promoter, Geminin stabilizes Cdt1 and facilitates its accumulation leading to the assembly of the pre-replication complex on DNA. As an inhibitor, Geminin prevents Cdt1 from loading the mini-chromosome maintenance complex onto pre-replication complexes in late S, G₂, and M phases. Here we show that during meiosis Geminin functions as a stabilizer of Cdt1 promoting its accumulation for the early division cycles of the embryo. Depletion of Geminin in Xenopus immature oocytes leads to a decrease of Cdt1 protein levels during maturation and after activation of these oocytes. Injection of exogenous recombinant Geminin into the depleted oocytes rescues Cdt1 levels demonstrating that Geminin stabilizes Cdt1 during meiosis and after fertilization. Furthermore, Geminin-depleted oocytes did not replicate their DNA after meiosis I indicating that Geminin does not act as an inhibitor of initiation of DNA replication between meiosis I and meiosis II.In eukaryotes, initiation of DNA replication involves the formation and activation of the pre-replication complex (pre-RC)³ at the origins of replication. Pre-RCs are formed by the sequential binding of the origin recognition complex components, Cdc6, Cdt1, and mini-chromosome maintenance complex (MCM 2–7) proteins, to DNA. After loading the MCM complex, the pre-RCs are activated by S phase kinases (Dbf4-dependent kinase and Cdks) to initiate DNA replication (1). Replication of DNA, limited to only once per cell cycle, is critical to maintain genomic stability. Redundant mechanisms exist to ensure that DNA replication is tightly regulated during the cell cycle (1, 2). A small protein named Geminin has been shown to play a significant role in such regulatory mechanisms during mitosis (2–6). Geminin, a multifunctional 25-kDa protein, was first identified in a screen for proteins degraded during mitosis in Xenopus laevis egg extracts (7). Geminin is present in higher eukaryotes, but its presence in yeast has not yet been reported (7–10). Geminin plays a major role in regulating the function of Cdt1, one of the pre-RC components (8, 11–13). Numerous studies suggest that in higher eukaryotes the interaction between Geminin and Cdt1 is pivotal to restrict DNA replication to only once per cell cycle (6, 14–22). Furthermore, in Xenopus egg extracts, the Geminin/Cdt1 ratio seems to control the assembly of pre-RCs at replication origins and to determine whether the origins are licensed or not (23). The positive and negative roles of Geminin in origin licensing and DNA replication are made possible by their temporal separation during the cell cycle. Pre-RC formation occurs during late M and early G₁ phase, whereas pre-RC inhibition occurs from late S to mid M phase.As a positive regulator of DNA replication, Geminin has been shown to stabilize Cdt1. In human osteosarcoma cells, silencing of GEMININ expression limits CDT1 accumulation during mitosis and therefore the formation of pre-RCs in the subsequent cell cycle. This stabilizing effect is the result of a direct interaction between CDT1 and GEMININ preventing CDT1 ubiquitination and degradation (13). Similar findings were also recently observed in normal human cells and various cancer cells (24). However, in both human normal and tumor cells, the low level of CDT1, generated by the absence of GEMININ, did not always prevent cellular proliferation or re-replication of the genome (5, 24, 25). Therefore, one might question the importance of the role of GEMININ in stabilizing CDT1 in human cells. Beyond its role as a stabilizer of Cdt1 levels, Geminin has also been shown to participate directly in the formation of pre-RCs in Xenopus egg extracts. A complex between Cdt1 and Geminin binds to chromatin and supports pre-RC assembly. However, the recruitment of additional Geminin molecules to this complex on the chromatin blocks further pre-RC formation. These results indicate that the stoichiometry of Cdt1 and Geminin in this complex regulates its activity as a promoter or inhibitor of pre-RC assembly and DNA replication (23, 26). Several mechanisms have been shown to modulate the Geminin/Cdt1 balance on the chromatin. In Xenopus the binding of Cdt1 to the MCM9 protein seems to block the recruitment of an excess of Geminin to the chromatin and therefore favors pre-RC assembly (27). Similarly, the inactivation of Geminin by either ubiquitination or degradation also has a positive effect on pre-RC assembly (8, 11, 28–30). On the other hand, the replication-dependent degradation of Cdt1 has the opposite effect and prevents refiring of replication origins during S and G₂ phases of the mitotic cell cycle (18, 20, 31).Although the role of Geminin during mitosis has been extensively studied, not much is known about its function during meiosis. The expression pattern of Geminin during oocyte maturation is unclear. The presence of Geminin in immature stage VI Xenopus oocytes is controversial, but the protein is fully expressed in mature oocytes arrested in metaphase of meiosis II (7, 32). To form haploid gametes, DNA replication has to be inhibited between meiosis I (MI) and meiosis II (MII). In Xenopus oocytes, cyclin B-dependent kinase 1 (Cdk1) also known as maturation-promoting factor (MPF) plays a role in preventing DNA replication between the two meiotic divisions (33–36). Inhibition of Cdk1 activity between MI and MII leads to the formation of interphase nucleus and DNA replication. However, the role of Geminin in preventing DNA replication between meiotic divisions has not been tested so far. Finally, the possibility that Geminin stabilizes Cdt1 during meiosis and ensures its accumulation for the early embryonic divisions has not been formally examined.Here we show that the levels of Geminin and Cdt1 proteins increase significantly during meiosis in Xenopus oocytes and that the primary role of geminin is to promote the accumulation of Cdt1 and not to repress DNA replication between meiosis I and meiosis II. Depletion of Geminin in Xenopus immature oocytes does not lead to DNA replication after the first meiotic division but to a decrease in Cdt1 stability during the maturation and activation of these oocytes. Rescue of Cdt1 levels in these Geminin-depleted oocytes is achieved by injection of exogenous recombinant Geminin protein confirming the role of Geminin as a stabilizer of Cdt1 during meiosis and the early embryonic division cycles. These results provide further support for the idea that Geminin functions universally in stabilizing Cdt1. Although the stabilizing role of Geminin might not be its most important function in somatic cells, we show here that stabilizing Cdt1 is a dominant function for Geminin in Xenopus oocytes undergoing meiosis. This stabilizing role of Geminin is essential for the stockpiling of Cdt1 before fertilization that is required to sustain the rapid divisions of the early embryo.     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.