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Genomic analysis and biological characterization of a novel Schitoviridae phage infecting Vibrio alginolyticus

Tajuddin S, Khan AM, Chong LC, Wong CL, Tan JS, Ina-Salwany MY, et al.

Appl Microbiol Biotechnol, 2023 Feb;107(2-3):749-768.
PMID: 36520169 DOI: 10.1007/s00253-022-12312-3

Vibrio alginolyticus is a Gram-negative bacterium commonly associated with mackerel poisoning. A bacteriophage that specifically targets and lyses this bacterium could be employed as a biocontrol agent for treating the bacterial infection or improving the shelf-life of mackerel products. However, only a few well-characterized V. alginolyticus phages have been reported in the literature. In this study, a novel lytic phage, named ΦImVa-1, specifically infecting V. alginolyticus strain ATCC 17749, was isolated from Indian mackerel. The phage has a short latent period of 15 min and a burst size of approximately 66 particles per infected bacterium. ΦImVa-1 remained stable for 2 h at a wide temperature (27-75 °C) and within a pH range of 5 to 10. Transmission electron microscopy revealed that ΦImVa-1 has an icosahedral head of approximately 60 nm in diameter with a short tail, resembling those in the Schitoviridae family. High throughput sequencing and bioinformatics analysis elucidated that ΦImVa-1 has a linear dsDNA genome of 77,479 base pairs (bp), with a G + C content of ~ 38.72% and 110 predicted gene coding regions (106 open reading frames and four tRNAs). The genome contains an extremely large virion-associated RNA polymerase gene and two smaller non-virion-associated RNA polymerase genes, which are hallmarks of schitoviruses. No antibiotic genes were found in the ΦImVa-1 genome. This is the first paper describing the biological properties, morphology, and the complete genome of a V. alginolyticus-infecting schitovirus. When raw mackerel fish flesh slices were treated with ΦImVa-1, the pathogen loads reduced significantly, demonstrating the potential of the phage as a biocontrol agent for V. alginolyticus strain ATCC 17749 in the food. KEY POINTS: • A novel schitovirus infecting Vibrio alginolyticus ATCC 17749 was isolated from Indian mackerel. • The complete genome of the phage was determined, analyzed, and compared with other phages. • The phage is heat stable making it a potential biocontrol agent in extreme environments.

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics
Fulltext Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Hassan MI, McSorley FR, Hotta K, Boddy CN

J Vis Exp, 2017 06 27.
PMID: 28715370 DOI: 10.3791/55187

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics*
Fulltext Impact of RNA Virus Evolution on Quasispecies Formation and Virulence

Mandary MB, Masomian M, Poh CL

Int J Mol Sci, 2019 Sep 19;20(18).
PMID: 31546962 DOI: 10.3390/ijms20184657

RNA viruses are known to replicate by low fidelity polymerases and have high mutation rates whereby the resulting virus population tends to exist as a distribution of mutants. In this review, we aim to explore how genetic events such as spontaneous mutations could alter the genomic organization of RNA viruses in such a way that they impact virus replications and plaque morphology. The phenomenon of quasispecies within a viral population is also discussed to reflect virulence and its implications for RNA viruses. An understanding of how such events occur will provide further evidence about whether there are molecular determinants for plaque morphology of RNA viruses or whether different plaque phenotypes arise due to the presence of quasispecies within a population. Ultimately this review gives an insight into whether the intrinsically high error rates due to the low fidelity of RNA polymerases is responsible for the variation in plaque morphology and diversity in virulence. This can be a useful tool in characterizing mechanisms that facilitate virus adaptation and evolution.

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics*
Development of a T7 RNA polymerase expressing cell line using lentivirus vectors for the recovery of recombinant Newcastle disease virus

Yeong MY, Cheow PS, Abdullah S, Song AA, Lei-Rossmann J, Tan TK, et al.

J Virol Methods, 2021 05;291:114099.
PMID: 33592218 DOI: 10.1016/j.jviromet.2021.114099

The development of a T7 RNA polymerase (T7 RNAP) expressing cell line i.e. BSR T7/5 cells marks an improvement of reverse genetics for the recovery of recombinant Newcastle disease virus (rNDV). BSR T7/5 is developed by transient transfection of plasmid encoding T7 RNAP gene for rNDV rescue. However, the gene expression decreases gradually over multiple passages and eventually hinders the rescue of rNDV. To address this issue, lentiviral vector was used to develop T7 RNAP-expressing HEK293-TA (HEK293-TA-Lv-T7) and SW620 (SW620-Lv-T7) cell lines, evidenced by the expression of T7 RNAP after subsequent 20 passages. rNDV was rescued successfully using HEK293-TA-Lv-T7 clones (R1D3, R1D8, R5B9) and SW620-Lv-T7 clones (R1C11, R3C5) by reverse transfection, yielding comparable virus rescue efficiency and virus titres to that of BSR T7/5. This study provides new tools for rNDV rescue and insights into cell line development and virology by reverse genetics.

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics
Fulltext Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis

Zaw MT, Emran NA, Lin Z

J Infect Public Health, 2018 04 26;11(5):605-610.
PMID: 29706316 DOI: 10.1016/j.jiph.2018.04.005

BACKGROUND: Rifampicin (RIF) plays a pivotal role in the treatment of tuberculosis due to its bactericidal effects. Because the action of RIF is on rpoB gene encoding RNA polymerase β subunit, 95% of RIF resistant mutations are present in rpoB gene. The majority of the mutations in rpoB gene are found within an 81bp RIF-resistance determining region (RRDR).
METHODOLOGY: Literatures on RIF resistant mutations published between 2010 and 2016 were thoroughly reviewed.
RESULTS: The most commonly mutated codons in RRDR of rpoB gene are 531, 526 and 516. The possibilities of absence of mutation in RRDR of rpoB gene in MDR-TB isolates in few studies was due to existence of other rare rpoB mutations outside RRDR or different mechanism of rifampicin resistance.
CONCLUSION: Molecular methods which can identify extensive mutations associated with multiple anti-tuberculous drugs are in urgent need so that the research on drug resistant mutations should be extended.

Matched MeSH terms: DNA-Directed RNA Polymerases
Molecular characterization of the polymerase gene and genomic termini of Nipah virus

Harcourt BH, Tamin A, Halpin K, Ksiazek TG, Rollin PE, Bellini WJ, et al.

Virology, 2001 Aug 15;287(1):192-201.
PMID: 11504554

In 1998, Nipah virus (NV) emerged in peninsular Malaysia, causing fatal encephalitis in humans and a respiratory disease in swine. NV is most closely related to Hendra virus (HV), a paramyxovirus that was identified in Australia in 1994, and it has been proposed that HV and NV represent a new genus within the family Paramyxoviridae. This report describes the analysis of the sequences of the polymerase gene (L) and genomic termini of NV as well as a comparison of the full-length, genomic sequences of HV and NV. The L gene of NV is predicted to be 2244 amino acids in size and contains the six domains found within the L proteins of all nonsegmented, negative-stranded (NNS) RNA viruses. However, the GDNQ motif found in most NNS RNA viruses was replaced by GDNE in both NV and HV. The 3' and 5' termini of the NV genome are nearly identical to the genomic termini of HV and share sequence homology with the genomic termini of other members of the subfamily Paramyxovirinae. At 18,246 nucleotides, the genome of NV is 12 nucleotides longer than the genome of HV and they have the largest genomes within the family Paramyxoviridae. The comparison of the structures of the genomes of HV and NV is now complete and this information will help to establish the taxonomic position of these novel viruses within the family Paramyxoviridae.

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics*; DNA-Directed RNA Polymerases/chemistry
Fulltext The multiple roles of hypothetical gene BPSS1356 in Burkholderia pseudomallei

Yam H, Rahim AA, Mohamad S, Mahadi NM, Manaf UA, Shu-Chien AC, et al.

PLoS One, 2014;9(6):e99218.
PMID: 24927285 DOI: 10.1371/journal.pone.0099218

Burkholderia pseudomallei is an opportunistic pathogen and the causative agent of melioidosis. It is able to adapt to harsh environments and can live intracellularly in its infected hosts. In this study, identification of transcriptional factors that associate with the β' subunit (RpoC) of RNA polymerase was performed. The N-terminal region of this subunit is known to trigger promoter melting when associated with a sigma factor. A pull-down assay using histidine-tagged B. pseudomallei RpoC N-terminal region as bait showed that a hypothetical protein BPSS1356 was one of the proteins bound. This hypothetical protein is conserved in all B. pseudomallei strains and present only in the Burkholderia genus. A BPSS1356 deletion mutant was generated to investigate its biological function. The mutant strain exhibited reduced biofilm formation and a lower cell density during the stationary phase of growth in LB medium. Electron microscopic analysis revealed that the ΔBPSS1356 mutant cells had a shrunken cytoplasm indicative of cell plasmolysis and a rougher surface when compared to the wild type. An RNA microarray result showed that a total of 63 genes were transcriptionally affected by the BPSS1356 deletion with fold change values of higher than 4. The expression of a group of genes encoding membrane located transporters was concurrently down-regulated in ΔBPSS1356 mutant. Amongst the affected genes, the putative ion transportation genes were the most severely suppressed. Deprivation of BPSS1356 also down-regulated the transcriptions of genes for the arginine deiminase system, glycerol metabolism, type III secretion system cluster 2, cytochrome bd oxidase and arsenic resistance. It is therefore obvious that BPSS1356 plays a multiple regulatory roles on many genes.

Matched MeSH terms: DNA-Directed RNA Polymerases/metabolism; DNA-Directed RNA Polymerases/chemistry
Performance comparison between bootstrap and multiscale bootstrap for assessing phylogenetic tree for RNA polymerase

Safinah Sharuddin, Nora Muda

Sains Malaysiana, 2015;44:1643-1651.

Phylogenetic inference refers to the reconstruction of evolutionary relationships among various species that is usually
presented in the form of a tree. This study constructs the phylogenetic tree by using a novel distance-based method known
as Modified one step M-estimator (MOM) method. The branches of the phylogenetic tree constructed were then evaluated
to see their reliability. The performance of the reliability was then compared between the p-value of multiscale bootstrap
(AU value) and bootstrap p-value (BP value). The aim of this study was to compare the performance between the AU value
and BP value for assessing phylogenetic tree of RNA polymerase. The results have shown that multiscale bootstrap analysis
can detect high sampling errors but not in bootstrap analysis. To overcome this problem, the multiscale bootstrap analysis
has reduced the sampling error by increasing the number of replications. The clusters were indicated as significant if AU
values or BP values were 95% or higher. From the analysis, the results showed that the BP and AU values differ at 11th
and 15th branch of the phylogenetic tree. The BP values at both branches were 72 and 85%, respectively, thereby making
the cluster not significant but by looking at the AU values, the two branches were more than 95% and the clusters were
significant. This was due to the biasness in calculation of the probability of bootstrap analysis, therefore, the multiscale
bootstrap analysis has improved the calculation of the probability value compared to the bootstrap analysis.

Matched MeSH terms: DNA-Directed RNA Polymerases
Global metabolic network reorganization by adaptive mutations allows fast growth of Escherichia coli on glycerol

Cheng KK, Lee BS, Masuda T, Ito T, Ikeda K, Hirayama A, et al.

Nat Commun, 2014;5:3233.
PMID: 24481126 DOI: 10.1038/ncomms4233

Comparative whole-genome sequencing enables the identification of specific mutations during adaptation of bacteria to new environments and allelic replacement can establish their causality. However, the mechanisms of action are hard to decipher and little has been achieved for epistatic mutations, especially at the metabolic level. Here we show that a strain of Escherichia coli carrying mutations in the rpoC and glpK genes, derived from adaptation in glycerol, uses two distinct metabolic strategies to gain growth advantage. A 27-bp deletion in the rpoC gene first increases metabolic efficiency. Then, a point mutation in the glpK gene promotes growth by improving glycerol utilization but results in increased carbon wasting as overflow metabolism. In a strain carrying both mutations, these contrasting carbon/energy saving and wasting mechanisms work together to give an 89% increase in growth rate. This study provides insight into metabolic reprogramming during adaptive laboratory evolution for fast cellular growth.

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics*
Phylogenetic designation of enterovirus 71 genotypes and subgenotypes using complete genome sequences

Chan YF, Sam IC, AbuBakar S

Infect Genet Evol, 2010 Apr;10(3):404-12.
PMID: 19465162 DOI: 10.1016/j.meegid.2009.05.010

Human enterovirus 71 (EV-71) is genotyped for molecular epidemiological investigation mainly using the two structural genes, VP1 and VP4. Based on these, EV-71 is divided into three genotypes, A, B and C, and within the genotypes B and C, there are further subgenotypes, B1-B5 and C1-C5. Classification using these genes is useful but gives incomplete phylogenetic information. In the present study, the phylogenetic relationships amongst all the known EV-71 and human enterovirus A (HEV-A) isolates with complete genome sequences were examined. A different tree topology involving EV-71 isolates of subgenotypes, C4 and B5 was obtained in comparison to that drawn using VP1. The nucleotide sequence divergence of the C4 isolates was 18.11% (17-20%) when compared to other isolates of subgenotype C. However, this positions the C4 isolates within the cut-off divergence value of 17-22% used to designate the virus genotypes. Hence, it is proposed here that C4 should be designated as a new genotype D. In addition, the subgenotype B5 isolates had an average nucleotide divergence of only 6.14% (4-8%) when compared to other subgenotype B4 isolates. This places the B5 isolates within the subgenotype B4. It is proposed here that the B5 isolates to be redesignated as B4. With the newly proposed genotype D and inclusion of subgenotype B5 within B4, the average nucleotide divergence between genotypes was 18.99% (17-22%). Inter- and intra-subgenotype average divergences were 12.02% (10-14%) and 3.92% (1-10%), respectively. A phylogenetic tree built using the full genome sequences is robust as it takes into consideration changes in the sequences of both the structural and non-structural genes. Similar nucleotide similarities, however, were obtained if only VP1 and 3D RNA polymerase genes were used. Furthermore, addition of 3D RNA polymerase sequences will also show recombination events. Hence, in the absence of full genome sequences, it is proposed here that a combination of VP1 and 3D RNA polymerase gene sequences be used for initial genotyping of EV-71 isolates.

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics
RpoS differentially affects the general stress response and biofilm formation in the endophytic Serratia plymuthica G3

Liu X, Wu Y, Chen Y, Xu F, Halliday N, Gao K, et al.

Res. Microbiol., 2016 Apr;167(3):168-77.
PMID: 26671319 DOI: 10.1016/j.resmic.2015.11.003

The σ(S) subunit RpoS of RNA polymerase functions as a master regulator of the general stress response in Escherichia coli and related bacteria. RpoS has been reported to modulate biocontrol properties in the rhizobacterium Serratia plymuthica IC1270. However, the role of RpoS in the stress response and biofilm formation in S. plymuthica remains largely unknown. Here we studied the role of RpoS from an endophytic S. plymuthica G3 in regulating these phenotypes. Mutational analysis demonstrated that RpoS positively regulates the global stress response to acid or alkaline stresses, oxidative stress, hyperosmolarity, heat shock and carbon starvation, in addition to proteolytic and chitinolytic activities. Interestingly, rpoS mutations resulted in significantly enhanced swimming motility, biofilm formation and production of the plant auxin indole-3-acetic acid (IAA), which may contribute to competitive colonization and environmental fitness for survival. These findings provide further insight into the strain-specific role of RpoS in the endophytic strain G3 of S. plymuthica, where it confers resistance to general stresses encountered within the plant environment. The heterogeneous functionality of RpoS in rhizosphere and endophytic S. plymuthica populations may provide a selective advantage for better adaptation to various physiological and environmental stresses.

Matched MeSH terms: DNA-Directed RNA Polymerases
Fulltext Comparative analysis of ribosomal protein gene, el14 expression between two types of colorectal carcinoma cell lines

Melinda, Mei Lin Lau, Edmund, Sim Ui Hang

Trends in Undergraduate Research, 2020;3(1):13-17.
MyJurnal

Association between the expression of ribosomal protein (RP) genes and cancer is widely known. More specifically, the extra-ribosomal functions of RPs have been linked to carcinogenesis. The ribosomal protein gene, eL14 has been reported to be associated with malignancy of the colorectum, albeit of mechanism yet unclear. Its expression in cells derived from different tissue origin of colorectal carcinoma (CRC) has never been explored. Therefore, this study aims to comparatively analyse the expression pattern of eL14 between two different CRC cell lines (DLD-1 and HCT116). It involved a conventional gene expression analysis, the Reverse-Transcriptase PCR (RT-PCR) assays. Products of RT-PCR assay were resolved via an agarose gel electrophoresis method, and band intensities of amplicons were documented and quantified using TotalLab Quant software. We observed differential expression patterns of eL14 between DLD-1 and HCT116 cells, but statistical analysis revealed insignificant differences. Therefore, the relevance of eL14 as a biomarker to distinguish between different colorectal cancer cells is suggestive but not conclusive.

Matched MeSH terms: DNA-Directed RNA Polymerases
Fulltext Phenotypic and Genetic Diversity of Aeromonas Species Isolated from Fresh Water Lakes in Malaysia

Khor WC, Puah SM, Tan JA, Puthucheary SD, Chua KH

PLoS One, 2015;10(12):e0145933.
PMID: 26710336 DOI: 10.1371/journal.pone.0145933

Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions--exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics
Lactobacillus nasalidis sp. nov., isolated from the forestomach of a captive proboscis monkey (Nasalis larvatus)

Suzuki-Hashido N, Tsuchida S, Hayakawa T, Sakamoto M, Azumano A, Seino S, et al.

Int J Syst Evol Microbiol, 2021 Apr;71(4).
PMID: 33906706 DOI: 10.1099/ijsem.0.004787

Three strains (YZ01T, YZ02 and YZ03) of Gram-stain-positive, facultatively anaerobic rods were isolated from the forestomach contents collected from a captive male proboscis monkey (Nasalis larvatus) at Yokohama Zoo in Japan. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belonged to the genus Lactobacillus. Based on the sequence similarity of the 16S rRNA gene, Lactobacillus delbrueckii subsp. indicus JCM 15610T was the closest phylogenetic neighbour to YZ01T. Sequence analyses of two partial concatenated housekeeping genes, the RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase alpha subunit (pheS) also indicated that the novel strains belonged to the genus Lactobacillus. The average nucleotide identity and digital DNA-DNA hybridization (dDDH) between L. delbrueckii subsp. indicus and YZ01T were 85.9 and 31.4 %, respectively. The phylogenetic tree based on the whole genomic data of strains YZ01T, YZ02 and YZ03 suggested that these three strains formed a single monophyletic cluster in the genus Lactobacillus, indicating that it belonged to a new species. The DNA G+C content of strain YZ01T was 51.6 mol%. The major fatty acids were C16 : 0 and C18 : 1 ω9c. Therefore, based on phylogenetic, phenotypic and physiological evidence, strains YZ01T, YZ02 and YZ03 represent a novel species of the genus Lactobacillus, for which the name Lactobacillus nasalidis sp. nov. is proposed with the type strain YZ01T (=JCM 33769T=DSM 110539T).

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics
Pseudomonas versuta sp. nov., isolated from Antarctic soil

See-Too WS, Salazar S, Ee R, Convey P, Chan KG, Peix Á

Syst Appl Microbiol, 2017 Jun;40(4):191-198.
PMID: 28501448 DOI: 10.1016/j.syapm.2017.03.002

In this study we analysed three bacterial strains coded L10.10T, A4R1.5 and A4R1.12, isolated in the course of a study of quorum-quenching bacteria occurring in Antarctic soil. The 16S rRNA gene sequence was identical in the three strains and showed 99.7% pairwise similarity with respect to the closest related species Pseudomonas weihenstephanensis WS4993T. Therefore, the three strains were classified within the genus Pseudomonas. Analysis of housekeeping genes (rpoB, rpoD and gyrB) sequences showed similarities of 84-95% with respect to the closest related species of Pseudomonas, confirming its phylogenetic affiliation. The ANI values were less than 86% to the closest related species type strains. The respiratory quinone is Q9. The major fatty acids are C16:0, C16:1 ω7c/ C16:1 ω6c in summed feature 3 and C18:1 ω7c / C18:1 ω6c in summed feature 8. The strains are oxidase- and catalase-positive. Growth occurs at 4-30°C, and at pH 4.0-10. The DNA G+C content is 58.2-58.3mol %. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strains L10.10T, A4R1.5 and A4R1.12 into a novel species of Pseudomonas, for which the name P. versuta sp. nov. is proposed. The type strain is L10.10T (LMG 29628T, DSM 101070T).

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics
Application of multilocus sequence analysis for molecular characterization of enterococci with virulence factors recovered from a tropical recreational beach

Ahmad A, Dada AC, Usup G

Southeast Asian J. Trop. Med. Public Health, 2014 May;45(3):700-18.
PMID: 24974655

Partial gene sequences of phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) were evaluated for species delineation and detection of recombination among enterococci populations recovered from a bathing beach impacted by low tide river flow. At inter-species level, a maximum similarity of 86.5% and 94.8% was observed among the enterococci pheS and rpoA sequence, respectively. A superimposed plot of delimited pairwise similarity values obtained for 266 pair-wise observations revealed that while there was a harmony between species identity obtained from both genes, pheS was more discriminatory than rpoA. The difference was more pronounced for inter-species comparison. A number of putative recombination events between indigenous and non-indigenous strains was detected based on a library of aligned sequences. Virulence genes cyl, esp, gelE and asa were detected in 7, 22, 100 and 63%, respectively among river isolates but at lower proportion of 0, 20, 67 and 42%, respectively among beach water isolates. Random amplified polymorphic DNA profiling presented evidence suggesting low tide river as a source of fecal enterococci entering the recreation beach water. Multilocus sequence typing analysis of a number of Enterococcus faecalis isolates presented four sequence types, ST59, 117, 181 and 474. The presence of genetically diverse fecal enterococci with associated virulence traits and a background of recombination events in surface recreational water could present a potential public health risk.

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics*
Fulltext Isolation and molecular identification of Bartonellae from wild rats (Rattus species) in Malaysia

Tay ST, Mokhtar AS, Zain SN, Low KC

Am J Trop Med Hyg, 2014 Jun;90(6):1039-42.
PMID: 24732465 DOI: 10.4269/ajtmh.13-0273

This study describes our investigation on the prevalence and molecular identification of bartonellae from Rattus diardii and R. norvegicus in the urban areas of Malaysia. Of 95 rats investigated, Bartonella tribocorum, B. rattimassiliensis, B. coopersplainsensis, B. elizabethae, and B. queenslandensis were isolated from kidney and spleen homogenates of four rats. Bartonellae DNA was amplified from the rat organ tissues by using primers specific for the bartonellae RNA polymerase beta subunit (rpoB) gene in nine other rats. Sequence analysis of the rpoB gene fragments shows the identification of B. queenslandensis in five rats, B. elizabethae in three rats, and B. tribocorum in one rat. Combining the results of isolation and molecular detection of bartonellae, we found that the prevalence of Bartonella infection in the Rattus spp. investigated in this study was 13.7%. Implementation of effective rat control program in the urban areas is necessary to prevent the spillover of bartonellosis from rats to humans.

Matched MeSH terms: DNA-Directed RNA Polymerases/genetics
Genome structure of Sagiyama virus and its relatedness to other alphaviruses

Shirako Y, Yamaguchi Y

J Gen Virol, 2000 May;81(Pt 5):1353-60.
PMID: 10769079

Sagiyama virus (SAG) is a member of the genus Alphavirus in the family Togaviridae, isolated in Japan from mosquitoes in 1956. We determined the complete nucleotide sequence of the SAG genomic RNA from the original stock virus which formed a mixture of plaques with different sizes, and that from a full-length cDNA clone, pSAG2, infectious RNA transcripts from which formed uniform large plaques on BHK-21 cells. The SAG genome was 11698 nt in length exclusive of the 3' poly(A) tail. Between the complete nucleotide sequences of the full-length cDNA clone, pSAG2, and the consensus sequence from the original stock virus, there were nine amino acid differences; two each in nsP1, nsP2 and E1, and three in E2, some of which may be responsible for plaque phenotypic variants in the original virus stock. SAG was most closely related to Ross River virus among other alphaviruses fully sequenced, with amino acid sequence identities of 86% in the nonstructural proteins and of 83% in the structural proteins. The 3' terminal 280 nt region of SAG was 82% identical to that of Barmah Forest virus, which was otherwise not closely related to SAG. Comparison of the nucleotide sequence of SAG with partial nucleotide sequences of Getah virus (GET), which was originally isolated in Malaysia in 1955 and is closely related to SAG in serology and in biology, showed near identity between the two viruses, suggesting that SAG is a strain of GET.

Matched MeSH terms: DNA-Directed RNA Polymerases*
Fulltext Transcriptome analysis of Streptococcus pneumoniae treated with the designed antimicrobial peptides, DM3

Le CF, Gudimella R, Razali R, Manikam R, Sekaran SD

Sci Rep, 2016 05 26;6:26828.
PMID: 27225022 DOI: 10.1038/srep26828

In our previous studies, we generated a short 13 amino acid antimicrobial peptide (AMP), DM3, showing potent antipneumococcal activity in vitro and in vivo. Here we analyse the underlying mechanisms of action using Next-Generation transcriptome sequencing of penicillin (PEN)-resistant and PEN-susceptible pneumococci treated with DM3, PEN, and combination of DM3 and PEN (DM3PEN). DM3 induced differential expression in cell wall and cell membrane structural and transmembrane processes. Notably, DM3 altered the expression of competence-induction pathways by upregulating CelA, CelB, and CglA while downregulating Ccs16, ComF, and Ccs4 proteins. Capsular polysaccharide subunits were downregulated in DM3-treated cells, however, it was upregulated in PEN- and DM3PEN-treated groups. Additionally, DM3 altered the amino acids biosynthesis pathways, particularly targeting ribosomal rRNA subunits. Downregulation of cationic AMPs resistance pathway suggests that DM3 treatment could autoenhance pneumococci susceptibility to DM3. Gene enrichment analysis showed that unlike PEN and DM3PEN, DM3 treatment exerted no effect on DNA-binding RNA polymerase activity but observed downregulation of RpoD and RNA polymerase sigma factor. In contrast to DM3, DM3PEN altered the regulation of multiple purine/pyrimidine biosynthesis and metabolic pathways. Future studies based on in vitro experiments are proposed to investigate the key pathways leading to pneumococcal cell death caused by DM3.

Matched MeSH terms: DNA-Directed RNA Polymerases
Fulltext Citrus bent leaf viroid

Khoo, Ying Wei, Iftikhar, Yasir, Kong, Lih Ling, Ganesan Vadamalai

The Pertanika Journal of Scholarly Research Reviews, 2017;3(3):31-40.
MyJurnal

Citrus bent leaf viroid (CBLVd) from genus Apscaviroid, is one of the widely distributed viroids among the seven citrus viroids. It is comprised of three variants: Citrus viroid-Ia (CVd-Ia) (327 - 329 nucleotides), Citrus viroid-Ib (CVd-Ib) (315 - 319 nucleotides) and Citrus viroid-I-low sequence similarity (CVd-I-LSS) (325 - 330 nucleotides). Virulence of CBLVd totally expressed on citrus plants. Etrog citron (Citrus medica (L.)) coinfected with CBLVd, Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III) and Citrus viroid-V (CVd-V) showed epinasty, leaf rolling, and stunting. CBLVd has been reported to reduce the canopy proportion and fruit production of citrus trees inserted on trifoliate orange rootstock. Moreover, citrus tree infected with singly CBLVd or in combinations with CEVd, Hop stunt viroid (CVd-II) and CVd-III induced dwarfing have been associated with poor development of the root system. Reverse-transcriptase polymerase chain reaction (RT-PCR) amplification and multiplex reverse-transcriptase polymerase chain reaction (MRT-PCR) amplification have been widely used to detect citrus viroids including CBLVd. As citrus viroids are emerging threats in citrus groves, therefore, this review covers the evolution, geographical distribution and epidemiology, economic impact and symptomatology, host range and transmission, detection, and management will be helpful in formulating the integrated management strategies for CBLVd.

Matched MeSH terms: DNA-Directed RNA Polymerases

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