Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

ABSTRACT

We isolated a Vibrio fluvialis strain (IDH5335) from a stool sample collected from a patient with diarrhea. In this announcement, we report the complete genomic sequence of this organism, which was obtained by combining Illumina and Oxford Nanopore sequencing data.

DOI： 10.1128/mra.00707-23

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Strain KK2020170T, a Gram-stain negative, yellow colony-forming bacterium, was isolated from surface seawater sampled in Kojima Bay, Okayama, Japan. Phylogenetic analysis based on the 16S rRNA gene revealed that strain KK2020170T belongs to the genus Flavobacterium, with Flavobacterium haoranii LQY-7T (98.1% similarity) being its closest relative, followed by Flavobacterium sediminis MEBiC07310T (96.9%) and Flavobacterium urocaniciphilum YIT 12746T (96.0%). Whole-genome shotgun sequencing showed that strain KK2020170T, when paralleled with F. haoranii LQY-7 T, had 81.3% average nucleotide identity, and 24.6% in silico DNA-DNA hybridization values, respectively. The DNA G + C content of strain KK2020170T was 31.1 mol%. The most abundant fatty acids (> 10%) of strain KK2020170T were iso-C15: 0, iso-C17: 0 3-OH and iso-C15: 1 G. The dominant respiratory quinone of the strain was menaquinone MK-6. Based on the phylogenetic and phenotypic analysis results, we propose that strain KK2020170T represents a novel species, for which the name Flavobacterium okayamense sp. nov. has been proposed. The type strain is KK2020170T (= ATCC TSD-280 T = NBRC 115344 T).

DOI： 10.1007/s00203-023-03682-x

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.imlet.2023.09.009

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BACKGROUND: Southeast Asia is attractive for tourism. Unfortunately, travelers to this region are at risk of becoming infected with Shigella. We conducted a meta-analysis to provide updates on Shigella prevalence in Southeast Asia, along with their serogroups and serotypes. METHODS: We conducted a systematic search using PubMed, EMBASE, and Web of Science for peer-reviewed studies from 2000 and November 2022. We selected studies that detected Shigella in stools by culture or polymerase chain reaction (PCR). Two reviewers extracted the data using a standardized form and performed quality assessments using the Joanna Briggs Institute checklist. The random effects model was used to estimate the pooled prevalence of Shigella. RESULTS: During our search, we identified 4376 studies. 29 studies (from six Southeast Asian countries) were included in the systematic review, 21 each in the meta-analysis of the prevalence of Shigella (Sample size:109545) and the prevalence of Shigella serogroups. The pooled prevalence of Shigella was 4% (95% CI: 4-5%) among diarrhea cases. Shigella sonnei was the most abundant serogroup in Thailand (74%) and Vietnam (57%), whereas Shigella flexneri was dominant in Indonesia (72%) and Cambodia (71%). Shigella dysenteriae and Shigella boydii were uncommon (pooled prevalence of 1% each). The pooled prevalence of Shigella was 5% (95% CI: 4-6%) in children aged <5 years. The pooled prevalence showed a decreasing trend comparing data collected between 2000 and 2013 (5%; 95% CI: 4-6%) and between 2014 and 2022 (3%; 95% CI: 2-4%). Shigella prevalence was 6% in studies that included participants with mixed pathogens versus 3% in those without. Shigella flexneri serotype 2a was the most frequently isolated (33%), followed by 3a (21%), 1b (10%), 2b (3%), and 6 (3%). CONCLUSIONS: This study provides compelling evidence for the development of effective Shigella vaccines for residents of endemic regions and travellers to these areas.

DOI： 10.1016/j.tmaid.2023.102554

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.envres.2023.115374

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BACKGROUND: Shigella remains one of the most common causes of diarrhoea in South Asia. Current estimates of the prevalence of Shigella are critical for guiding control measures. We estimated the prevalence of Shigella species and serogroups in South Asia. METHODS: We performed a systematic review using PubMed, EMBASE, Google Scholar, and Web of Science for peer-reviewed studies published between 2000 and June 19, 2022. We also manually searched the reference lists of the reviewed studies to identify additional studies. We included studies that detected the presence of Shigella in stool by culture or polymerase chain reaction (PCR). Studies associated with outbreaks were excluded. Two investigators independently reviewed the studies, extracted the data, and performed quality assessment. A random-effects meta-analysis was performed to determine the pooled prevalence of Shigella. RESULTS: Our search yielded 5707 studies, of which 91 studies from five South Asian countries were included in the systematic review, 79 in the meta-analysis of Shigella prevalence and 63 in the meta-analysis of Shigella serogroups prevalence. The pooled prevalence of Shigella was 7% (95% CI: 6-7%), with heterogeneity (I2 = 98.7; p < 0.01). The prevalence of Shigella was higher in children aged < 5 years (10%; 95% CI: 8-11%), in rural areas (12%; 95% CI: 10-14%), and in studies using PCR (15%; 95% CI: 11-19%). Shigella flexneri (58%) was the most abundant serogroup, followed by Shigella sonnei (19%), Shigella boydii (10%), and Shigella dysenteriae (9%). S. flexneri 2a was the most frequently isolated serotype (36%), followed by serotype 3a (12%), serotype 6 (12%), and serotype 1b (6%). The prevalence of non-typeable Shigella was 10.0%. CONCLUSIONS: Although the prevalence of Shigella in South Asia remains generally high, it varies by age group and geographical area, with data lacking in some countries. Effective Shigella vaccines would be advantageous for both endemic communities and travellers.

DOI： 10.1093/jtm/taac132

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BACKGROUND: India is an attractive destination for travelers. Unfortunately, numerous reports exist on traveler's diarrhea (TD) and fecal colonization with extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) among international travelers visiting India. Here, we systematically reviewed studies published on the acquisition of ESBL-EC and TD attack rates among international visitors to India. METHODS: Design: Systematic review and meta-analysis. A systematic search was performed using Google Scholar, PubMed, EMBASE, Web of Science, and gray literature from 2000 to December 2021, for studies containing data for ESBL-EC acquisition or TD experience related to a trip to India. Random effects models were used to compute the prevalence of ESBL-EC acquisition and TD attack. RESULTS: The literature search yielded a total of 5023 records. Of these, 31 met our inclusion criteria for systematic review and only 17 could be meta-analyzed (9 for TD, and 8 for ESBL-EC). The overall pooled attack rate of TD was 39% (95% confidence interval, CI: 25-53%). In studies where travelers' memory was used to diagnose TD, the pooled attack rate of TD was slightly higher (42%, 95% CI: 21-64%) compared to those where TD was objectively documented (33%, 95% CI: 17-49%). There were significant risks to be colonized with ESBL-EC among the travelers who experienced TD. The pooled rate of ESBL-EC colonization was 72% (CI: 67-78%). Most ESBL-EC produced CTX-M-15 enzyme. Furthermore, most of the travelers who acquired ESBL-EC were from highly industrialized countries recruited from travel clinics: Canada (n = 80), Germany (n = 69), Netherlands (n = 20), Sweden (n = 18), Japan (n = 10), Finland (n = 8), USA (n = 7), Spain (n = 5), and Denmark (n = 3). CONCLUSIONS: TD pooled attack rate and ESBL-EC acquisition among international travelers visiting India were high in this study. However, we cannot make generalizations based upon this TD pooled attack rate for the current situation, due to a lack of current data. Our study highlights that travelers should be advised on TD to ensure that they do not disregard the risk of contracting TD and be better prepared as a result. It also illustrates the importance of international travel in acquiring antibiotic-resistant Escherichia coli.

DOI： 10.1186/s40794-022-00179-1

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OBJECTIVE: To evaluate the clinical accuracy of rapid diagnostic tests for the detection of Ebola virus. METHODS: We searched MEDLINE®, Embase® and Web of Science for articles published between 1976 and October 2021 reporting on clinical studies assessing the performance of Ebola virus rapid diagnostic tests compared with reverse transcription polymerase chain reaction (RT-PCR). We assessed study quality using the QUADAS-2 criteria. To estimate the pooled sensitivity and specificity of these rapid diagnostic tests, we used a bivariate random-effects meta-analysis. FINDINGS: Our search identified 113 unique studies, of which nine met the inclusion criteria. The studies were conducted in the Democratic Republic of the Congo, Guinea, Liberia and Sierra Leone and they evaluated 12 rapid diagnostic tests. We included eight studies in the meta-analysis. The pooled sensitivity and specificity of the rapid tests were 86% (95% confidence interval, CI: 80-91) and 95% (95% CI: 91-97), respectively. However, pooled sensitivity decreased to 83% (95% CI: 77-88) after removing outliers. Pooled sensitivity increased to 90% (95% CI: 82-94) when analysis was restricted to studies using the RT-PCR from altona Diagnostics as gold standard. Pooled sensitivity increased to 99% (95% CI: 67-100) when the analysis was restricted to studies using whole or capillary blood specimens. CONCLUSION: The included rapid diagnostic tests did not detect all the Ebola virus disease cases. While the sensitivity and specificity of these tests are moderate, they are still valuable tools, especially useful for triage and detecting Ebola virus in remote areas.

DOI： 10.2471/BLT.21.287496

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BACKGROUND: Approximately 2.9 million people worldwide suffer from cholera each year, many of whom are destitute. However, understanding of immunity against cholera is still limited. Several studies have reported the duration of antibodies following cholera; however, systematic reviews including a quantitative synthesis are lacking. OBJECTIVE: To meta-analyze cohort studies that have evaluated vibriocidal, cholera toxin B subunit (CTB), and lipopolysaccharide (LPS) antibody levels following a clinical cholera case. METHODS: Design: Systematic review and meta-analysis. We searched PubMed and Web of science for studies assessing antibodies against Vibrio cholerae in cohorts of patients with clinical cholera. Two authors independently extracted data and assessed the quality of included studies. Random effects models were used to pool antibody titers in adults and older children (aged ≥ 6 years). In sensitivity analysis, studies reporting data on young children (2-5 years) were included. RESULTS: Nine studies met our inclusion criteria for systematic review and seven for meta-analysis. The pooled mean of vibriocidal antibody titers in adults and older children (aged ≥ 6 years) was 123 on day 2 post-symptom onset, which sharply increased on day 7 (pooled mean = 6956) and gradually waned to 2247 on day 30, 578 on day 90, and 177 on day 360. Anti-CTB IgA antibodies also peaked on day 7 (pooled mean = 49), followed by a rapid decrease on day 30 (pooled mean = 21), and further declined on day 90 (pooled mean = 10), after which it plateaued from day 180 (pooled mean = 8) to 360 (pooled mean = 6). Similarly, anti-CTB IgG antibodies peaked in early convalescence between days 7 (pooled mean = 65) and 30 (pooled mean = 69), then gradually waned on days 90 (pooled mean = 42) and 180 (pooled mean = 30) and returned to baseline on day 360 (pooled mean = 24). Anti-LPS IgA antibodies peaked on day 7 (pooled mean = 124), gradually declined on day 30 (pooled mean = 44), which persisted until day 360 (pooled mean = 10). Anti LPS IgG antibodies peaked on day 7 (pooled mean = 94). Thereafter, they decreased on day 30 (pooled mean = 85), and dropped further on days 90 (pooled mean = 51) and 180 (pooled mean = 47), and returned to baseline on day 360 (pooled mean = 32). Sensitivity analysis including data from young children (aged 2-5 years) showed very similar findings as in the primary analysis. CONCLUSIONS: This study confirms that serological antibody (vibriocidal, CTB, and LPS) titers return to baseline levels within 1 year following clinical cholera, i.e., before the protective immunity against subsequent cholera wanes. However, this decay should not be interpreted as waning immunity because immunity conferred by cholera against subsequent disease lasts 3-10 years. Our study provides evidence for surveillance strategies and future research on vaccines and also demonstrates the need for further studies to improve our understanding of immunity against cholera.

DOI： 10.3390/ijerph19127141

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Fecal contamination of water sources and open defecation have been linked to cholera outbreaks in India. However, a systematic review on the drivers responsible for these outbreaks has yet to be published. Here, we systematically review the published literature on cholera outbreaks in India between 2011 and 2020. We searched studies in English in three databases (MEDLINE, EMBASE, and Web of Science) and the Integrated Disease Surveillance Program that tracks cholera outbreaks throughout India. Two authors independently extracted data and assessed the quality of the included studies. Quantitative data on the modes of transmission reviewed in this study were assessed for any change over time between 2011-2015 and 2016-2020. Our search retrieved 10823 records initially, out of which 81 full-text studies were assessed for eligibility. Among these 81 studies, 20 were eligible for inclusion in this review. There were 565 reported outbreaks between 2011 and 2020 that led to 45,759 cases and 263 deaths. Outbreaks occurred throughout the year; however, they exploded with monsoons (June through September). In Tamil Nadu, a typical peak of cholera outbreaks was observed from December to January. Seventy-two percent (33,089/45,759) of outbreak-related cases were reported in five states, namely Maharashtra, West Bengal, Punjab, Karnataka, and Madhya Pradesh. Analysis of these outbreaks highlighted the main drivers of cholera including contaminated drinking water and food, inadequate sanitation and hygiene (including open defecation), and direct contact between households. The comparison between 2011-2015 and 2016-2020 showed a decreasing trend in the outbreaks that arose due to damaged water pipelines. Many Indians still struggle with open defecation, sanitation, and clean water access. These issues should be addressed critically. In addition, it is essential to interrupt cholera short-cycle transmission (mediated by households, stored drinking water and foodstuffs) during an outbreak. As cholera is associated with deprivation, socio-economic development is the only long-term solution.

DOI： 10.3390/ijerph19095738

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We sought to summarize knowledge, misconceptions, beliefs, and practices about Ebola that might impede the control of Ebola outbreaks in Africa. We searched Medline, EMBASE, CINAHL, and Google Scholar (through May 2019) for publications reporting on knowledge, attitudes, and practices (KAP) related to Ebola in Africa. In total, 14 of 433 articles were included. Knowledge was evaluated in all 14 articles, and they all highlighted that there are misconceptions and risk behaviors during an Ebola outbreak. Some communities believed that Ebola spreads through the air, mosquito bites, malice from foreign doctors, witchcraft, and houseflies. Because patients believe that Ebola was caused by witchcraft, they sought help from traditional healers. Some people believed that Ebola could be prevented by bathing with salt or hot water. Burial practices where people touch Ebola-infected corpses were common, especially among Muslims. Discriminatory attitudes towards Ebola survivors or their families were also prevalent. Some Ebola survivors were not accepted back in their communities; the possibility of being ostracized from their neighborhoods was high and Ebola survivors had to lead a difficult social life. Most communities affected by Ebola need more comprehensive knowledge on Ebola. Efforts are needed to address misconceptions and risk behaviors surrounding Ebola for future outbreak preparedness in Africa.

DOI： 10.3390/ijerph19084714

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BACKGROUND: Cholera is an acute diarrheal disease caused by Vibrio cholerae O1 or O139. Cholera rapid diagnostic tests (RDTs) are widely used to screen for cholera cases. However, their accuracy has not been systematically reviewed. OBJECTIVES: To evaluate the diagnostic accuracy of cholera RDTs. METHODS: Systematic review and meta-analysis. DATA SOURCES: Medline, EMBASE and Web of science through to November 2020; references of included studies and a technical guidance on cholera RDTs. This review is registered with PROSPERO (CRD42021233124). STUDY ELIGIBILITY CRITERIA: Cross-sectional studies comparing the performance of cholera RDTs either to stool culture or PCR. PARTICIPANTS: Individuals with clinically suspected cholera. DATA EXTRACTION: Two authors independently extracted data and assessed the quality using Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) criteria. RESULTS: Eighteen studies were included in the systematic review of which 17 were used for meta-analysis. Crystal VC was the most frequently used RDT (13 studies), followed by Cholkit and Institut Pasteur cholera dipstick (three studies each), SD Bioline (two studies), Artron (one study) and Smart (one study). Using direct testing (n = 12 627 specimens), the bivariate random-effects model yielded a pooled sensitivity and specificity of 91% (95% CI 87%-94%) and 80% (95% CI 74%-84%), respectively. However, through alkaline peptone water (APW) enrichment (n = 3403 specimens), the pooled sensitivity and specificity were 89% (95% CI 79%-95%) and 98% (95% CI 95%-99%), respectively. CONCLUSION: Cholera RDTs, especially when enriched with APW, have moderate sensitivity and specificity. Although less useful for clinical management, the current generation of RDTs have clear utility for surveillance efforts if used in a principled manner. Enrichment of stool specimens in APW before using cholera RDTs reduces the possibility of obtaining false-positive results, despite the few cholera cases that go undetected. It is noteworthy that APW-enriched cholera RDTs are not necessarily rapid tests, and are not listed in the Global Task Force on Cholera Control/WHO target product profile.

DOI： 10.1016/j.cmi.2021.08.027

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Rotavirus (RV) is the leading cause of acute gastroenteritis and watery diarrhea in children under 5 years accounting for high morbidity and mortality in countries with poor socioeconomic status. Although vaccination against RV has been implemented in more than 100 countries, the efficacy of vaccine has been challenged in low-income settings. The lack of any FDA-approved drug against RV is an additional concern regarding the treatment associated with rotavirus-induced infantile death. With the purpose for the discovery of anti-RV therapeutics, we assessed anti-rotaviral potential of quercetin, a well-characterized antioxidant flavonoid. In vitro study revealed that quercetin treatment resulted in diminished production of RV-SA11 (simian strain) viral particles in a concentration-dependent manner as estimated by the plaque assay. Consistent with this result, Western blot analysis also revealed reduced synthesis of viral protein in quercetin-treated RV-SA11-infected MA104 cells compared to vehicle (DMSO) treated controls. Not surprisingly, infection of other RV strains A5-13 (bovine strain) and Wa (Human strain) was also found to be abridged in the presence of quercetin compared to DMSO. The IC50 of quercetin against three RV strains ranges between 2.79 and 4.36 Mm, and S.I. index is greater than 45. Concurrent to the in vitro results, in vivo study in mice model also demonstrated reduced expression of viral proteins and viral titer in the small intestine of quercetin-treated infected mice compared to vehicle-treated infected mice. Furthermore, the result suggested anti-rotaviral activity of quercetin to be interferon-independent. Mechanistic study revealed that the antiviral action of quercetin is co-related with the inhibition of RV-induced early activation of NF-κB pathway. Overall, this study delineates the strong anti-RV potential of quercetin and also proposes it as future therapeutics against rotaviral diarrhea.

DOI： 10.3389/fmicb.2022.951716

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UNLABELLED: The rapid diagnosis of cholera contributes to adequate outbreak management. This meta-analysis assesses the diagnostic accuracy of cholera rapid tests (RDTs) to detect Vibrio cholerae O1. METHODS: Systematic review and meta-analysis. We searched four databases (Medline, EMBASE, Google Scholar, and Web of Science up to 8 September 2021) for studies that evaluated cholera RDTs for the detection of V. cholerae O1 compared with either stool culture or polymerase chain reaction (PCR). We assessed the studies' quality using the QUADAS-2 criteria. In addition, in this update, GRADE approach was used to rate the overall certainty of the evidence. We performed a bivariate random-effects meta-analysis to calculate the pooled sensitivity and specificity of cholera RDTs. RESULTS: Overall, 20 studies were included in this meta-analysis. Studies were from Africa (n = 11), Asia (n = 7), and America (Haiti; n = 2). They evaluated eight RDTs (Crystal VC-O1, Crystal VC, Cholkit, Institut Pasteur cholera dipstick, SD Bioline, Artron, Cholera Smart O1, and Smart II Cholera O1). Using direct specimen testing, sensitivity and specificity of RDTs were 90% (95% CI, 86 to 93) and 86% (95% CI, 81 to 90), respectively. Cholera Sensitivity was higher in studies conducted in Africa [92% (95% CI, 89 to 94)] compared with Asia [82% (95% CI, 77 to 87)]. However, specificity [83% (95% CI, 71 to 91)] was lower in Africa compared with Asia [90% (95% CI, 84 to 94)]. GRADE quality of evidence was estimated as moderate. CONCLUSIONS: Against culture or PCR, current cholera RDTs have moderate sensitivity and specificity for detecting Vibrio cholerae O1.

DOI： 10.3390/diagnostics11112095

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

We isolated the novel strain Vibrio rotiferianus AM7 from the shell of an abalone. In this article, we report the complete genome sequence of this organism, which was obtained by combining Oxford Nanopore long-read and Illumina short-read sequencing data.

DOI： 10.1128/MRA.01591-19

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Staphylococcus arlettae is one coagulase-negative species in the bacterial genus Staphylococcus Here, we describe the closed complete genome sequence of S. arlettae strain P2, which was obtained using a hybrid approach combining Oxford Nanopore long-read and Illumina MiSeq short-read sequencing data.

DOI： 10.1128/MRA.00696-19

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Our knowledge as to how bacteria acquire antibiotic resistance is still fragmented, especially for the ribosome-targeting drugs. In this study, with the aim of finding novel mechanisms that render bacteria resistant to the ribosome-targeting antibiotics, we developed a general method to systematically screen for antibiotic resistant 16 S ribosomal RNAs (rRNAs), which are the major target for multiple antibiotics (e.g. spectinomycin, tetracycline, and aminoglycosides), and identify point mutations therein. We used Escherichia coli ∆7, a null mutant of the rrn (ribosomal RNA) operons, as a surrogate host organism to construct a metagenomic library of 16 S rRNA genes from the natural (non-clinical) environment. The library was screened for spectinomycin resistance to obtain four resistant 16 S rRNA genes from non-E. coli bacterial species. Bioinformatic analysis and site-directed mutagenesis identified three novel mutations - U1183C (the first mutation discovered in a region other than helix 34), and C1063U and U1189C in helix 34 - as well as three well-described mutations (C1066U, C1192G, and G1193A). These results strongly suggest that uncharacterized antibiotic resistance mutations still exist, even for traditional antibiotics.

DOI： 10.1038/s41598-018-23474-4

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Publishing type：Research paper (scientific journal)   Publisher：Applied RNA Bioscience  

DOI： 10.1007/978-981-10-8372-3_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

The 16 S rRNA sequence has long been used uncritically as a molecular clock to infer phylogenetic relationships among prokaryotes without fully elucidating the evolutionary changes that this molecule undergoes. In this study, we investigated the functional evolvability of 16 S rRNA, using comparative RNA function analyses between the 16 S rRNAs of Escherichia coli (Proteobacteria) and Acidobacteria (78% identity, 334 nucleotide differences) in the common genetic background of E. coli. While the growth phenotype of an E. coli mutant harboring the acidobacterial gene was disrupted significantly, it was restored almost completely following introduction of a 16 S rRNA sequence with a single base-pair variation in helix 44; the remaining 332 nucleotides were thus functionally similar to those of E. coli. Our results suggest that 16 S rRNAs share an inflexible cradle structure formed by ribosomal proteins and have evolved by accumulating species-specific yet functionally similar mutations. While this experimental evidence suggests the neutral evolvability of 16 S rRNA genes and hence satisfies the necessary requirements to use the sequence as a molecular clock, it also implies the promiscuous nature of the 16 S rRNA gene, i.e., the occurrence of horizontal gene transfer among bacteria.

DOI： 10.1038/s41598-017-10214-3

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Other Link： http://www.nature.com/articles/s41598-017-10214-3

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Current methods used for phylogenetic classification of prokaryotes largely rely on the sequences of 16S rRNA genes that are ubiquitously present in the cell. Theoretical basis of this methodology is based on the assumption that 16S rRNA genes are only vertically inherited and are thus indigenous to each species. However, microbial genomic analysis has revealed the existence of prokaryotic species containing two types of rRNA (rrn) operons of seemingly different origins. It has also been reported that some bacteria contain 16S rRNA that are mosaics of sequences from multiple species. This suggests that horizontal gene transfer (HGT) occurred for 16S rRNA genes. In addition, a recent HGT experiment mimicking the natural HGT process has shown that a wide range of foreign 16S rRNA genes can be transferred into Escherichia coli, including those from different phylogenetic classes (with a minimum sequence identity of 80.9% to the Escherichia coli 16S rRNA gene). Thus, in contrast to the complexity hypothesis that states informational genes are rarely horizontally transferred between species, 16S rRNA is occasionally amenable to HGT. Results of the current method for rapid identification and classification of prokaryotes based on the 16S rRNA gene should thus be carefully analyzed and interpreted.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The bacterial ribosome consists of three rRNA molecules and 57 proteins and plays a crucial role in translating mRNA-encoded information into proteins. Because of the ribosome's structural and mechanistic complexity, it is believed that each ribosomal component coevolves to maintain its function. Unlike 5S rRNA, 16S and 23S rRNAs appear to lack mutational robustness, because they form the structural core of the ribosome. However, using Escherichia coli Delta 7 (null mutant of operons) as a host, we have recently shown that an active hybrid ribosome whose 16S rRNA has been specifically substituted with that from non-E. coli bacteria can be reconstituted in vivo. To investigate the mutational robustness of 16S rRNA and the structural basis for its functionality, we used a metagenomic approach to screen for 16S rRNA genes that complement the growth of E. coli Delta 7. Various functional genes were obtained from the Gammaproteobacteria and Betaproteobacteria lineages. Despite the large sequence diversity (80.9-99.0% identity with E. coli 16S rRNA) of the functional 16S rRNA molecules, the doubling times (DTs) of each mutant increased only modestly with decreasing sequence identity (average increase in DT, 4.6 s per mutation). The three-dimensional structure of the 30S ribosome showed that at least 40.7% (628/1,542) of the nucleotides were variable, even at ribosomal protein-binding sites, provided that the secondary structures were properly conserved. Our results clearly demonstrate that 16S rRNA functionality largely depends on the secondary structure but not on the sequence itself.

DOI： 10.1073/pnas.1213609109

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Modifications of rRNAs are clustered in functional regions of the ribosome. In Helix 74 of Escherichia coli 23S rRNA, guanosines at positions 2069 and 2445 are modified to 7-methylguanosine(m(7)G) and N-2-methylguanosine(m(2)G), respectively. We searched for the gene responsible for m(7)G2069 formation, and identified rlmL, which encodes the methyltransferase for m(2)G2445, as responsible for the biogenesis of m(7)G2069. In vitro methylation of rRNA revealed that rlmL encodes a fused methyltransferase responsible for forming both m(7)G2069 and m(2)G2445. We renamed the gene rlmKL. The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK. Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit. In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.

DOI： 10.1093/nar/gkr1287

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Ribonuclease (RNase) T2 is involved in scavenging exogenous RNAs in the periplasmic space of bacteria. In Escherichia coli, although the 30S ribosomal subunit has long been known as a specific inhibitor of RNase T2 (designated as RNase I in E. coli), both the biochemical mechanisms and physiological roles of this interaction remain to be elucidated. Here we show, by creating hybrid ribosomes and mutational studies, that helix 41 (h41) of the E. coli 16S ribosomal RNA has a crucial role in the specific inhibition of RNase I. Notably, h41-mutant strains exhibit a lower survival rate at stationary phase and severe cell lysis when the post-segregation killing protein SrnB is expressed. These phenotypic defects accompany significant RNA degradation caused by RNase I. Thus, h41 in 16S rRNA provides a physiological benefit for the host cells in coping with the potential cytotoxicity of RNase T2.

DOI： 10.1038/ncomms1553

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Ribosome biogenesis is coupled to the transcription of ribosomal (r)RNAs, which enables an ordered and hierarchical assembly of the ribosome starting with the 5&apos;-terminal domain. We constructed four circular permutants (CPs) of Escherichia coli rRNAs in which the original termini of 16S or 23S rRNAs were genetically connected, and new termini were created elsewhere within the same rRNAs (in helix 33 of 16S rRNA, or in helices 45, 63, and 78 of 23S rRNA). Unexpectedly, all CPs tested were able to rescue E coli strain Delta 7 prrn, which lacks all chromosomal rRNA operons. This result demonstrates that hierarchical assembly from the 5&apos;-terminal domain of both 16S and 23S rRNAs is not essential for ribosomal formation in the cell. However, severe growth defects of all CPs were found in the absence of the DEAD box RNA helicase deaD, indicating that DeaD assists in the efficient assembly of each subunit in the cell.

DOI： 10.1016/j.molcel.2009.05.014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The wobble base of Escherichia coli elongator tRNA(Met) is modified to N-4-acetylcytidine (ac(4)C), which is thought to ensure the precise recognition of the AUG codon by preventing misreading of near-cognate AUA codon. By employing genome-wide screen of uncharacterized genes in Escherichia coli ('ribonucleome analysis'), we found the ypfI gene, which we named tmcA (tRNA(Met) cytidine acetyltransferase), to be responsible for ac(4)C formation. TmcA is an enzyme that contains a Walker-type ATPase domain in its N-terminal region and an N-acetyltransferase domain in its C-terminal region. Recombinant TmcA specifically acetylated the wobble base of E. coli elongator tRNA(Met) by utilizing acetyl-coenzyme A (CoA) and ATP (or GTP). ATP/GTP hydrolysis by TmcA is stimulated in the presence of acetyl-CoA and tRNA(Met). A mutation study revealed that E. coli TmcA strictly discriminates elongator tRNA(Met) from the structurally similar tRNA(Ile) by mainly recognizing the C27-G43 pair in the anticodon stem. Our findings reveal an elaborate mechanism embedded in tRNA(Met) and tRNA(Ile) for the accurate decoding of AUA/AUG codons on the basis of the recognition of wobble bases by the respective RNA-modifying enzymes.

DOI： 10.1038/emboj.2008.154

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.

DOI： 10.1093/nar/gkm356

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