RNA Polymerase Beta Research Articles

Reassessment of the genetic basis of natural rifampin resistance in the genus Rickettsia.

Rickettsia, a genus of obligate intracellular bacteria, includes species that cause significant human diseases. This study challenges previous claims that the Leucine-973 residue in the RNA polymerase beta subunit is the primary determinant of rifampin resistance in Rickettsia. We investigated a previously untested Rickettsia species, R. lusitaniae, from the Transitional group and found it susceptible to rifampin, despite possessing the Leu-973 residue. Interestingly, we observed the conservation of this residue in several rifampin-susceptible species across most Rickettsia phylogenetic groups. Comparative genomics revealed potential alternative resistance mechanisms, including additional amino acid variants that could hinder rifampin binding and genes that could facilitate rifampin detoxification through efflux pumps. Importantly, the evolutionary history of Rickettsia genomes indicates that the emergence of natural rifampin resistance is phylogenetically constrained within the genus, originating from ancient genetic features shared among a unique set of closely related Rickettsia species. Phylogenetic patterns appear to be the most reliable predictors of natural rifampin resistance, which is confined to a distinct monophyletic subclade known as Massiliae. The distinctive features of the RNA polymerase beta subunit in certain untested Rickettsia species suggest that R. raoultii, R. amblyommatis, R. gravesii, and R. kotlanii may also be naturally rifampin-resistant species.

Reclassification of Aestuariicella albida as Pseudomaricurvus albidus comb. nov. and Aestuariicella hydrocarbonica as Pseudomaricurvus hydrocarbonicus comb. nov. Based on Comparative Genomics and Molecular Synapomorphies.

The genus Aestuariicella has been recently reclassified as a member of the family Cellvibrionaceae. However, the taxonomic position of the genus as a distinct member of the family has not been clarified. In the present study, we performed multilayered analyses anchored on genome sequences to clarify the relationship between the genera Aestuariicella and Pseudomaricurvus within the family Cellvibrionaceae. Phylogenetic analyses based on 16S rRNA gene, RNA polymerase beta subunit (RpoB) protein, and core gene sequences showed a well-supported tight cluster formed by the members of the two genera. Moreover, the analysis of the average amino acid identity (AAI) revealed that the members of the two genera shared 68.16-79.48% AAI, values which were within the range of observed AAI (≥ 67.23%) among the members of the same genus within the family Cellvibrionaceae. Members of the two genera also shared several common characteristics. Furthermore, molecular synapomorphies in a form of conserved signature indels were identified in six protein sequences that were exclusively shared by the members of the two genera. Based on the phylogenetic and molecular evidence presented here, we propose the reclassification of the species Aestuariicella albida and Aestuariicella hydrocarbonica as Pseudomaricurvus albidus comb. nov. and Pseudomaricurvus hydrocarbonicus comb. nov., respectively.

Molecular detection and characterization of Bartonella spp. in small mammals in the Amazonia and Cerrado biomes, midwestern Brazil

Although Bartonella spp. have been worldwide described in rodents and bats, few studies have reported these agents in marsupials. The present work aimed to investigate the occurrence and genetic diversity of Bartonella in small mammals (rodents, marsupials, and bats) and associated ectoparasites in two ecoregions (Amazonia and Cerrado biomes) in midwestern Brazil. For this purpose, DNA samples from 378 specimens of small mammals (128 rodents, 111 marsupials, and 139 bats) and 41 fleas (Siphonaptera) were screened for the Bartonella genus employing a quantitative real-time PCR assay (qPCR) based on the nuoG (nicotinamide adenine dinucleotide dehydrogenase gamma subunit) gene. Then, positive samples in qPCR were submitted to conventional PCR (cPCR) assays targeting the gltA, ftsZ, and rpoB genes. One (0.78 %) rodent, 23 (16.54 %) bats, and 3 (7.31 %) fleas showed positive results in the qPCR for Bartonella sp. After cPCR amplification and sequencing, 13 partial Bartonella DNA sequences of the following genes were obtained only from bats´ blood samples: 9 gltA (citrate synthase), 3 ftsZ (cell division protein), and 1 rpoB (RNA polymerase beta subunit). The maximum likelihood inference based on the gltA gene positioned the obtained sequences in three different clades, closely related to Bartonella genotypes previously detected in other bat species and bat flies sampled in Brazil and other countries from Latin America. Similarly, the ftsZ sequences clustered in two different clades with sequences described in bats from Brazil, other countries from Latin America, and Georgia (eastern Europe). Finally, the Bartonella rpoB from a specimen of Lophostoma silvicolum clustered with a Bartonella sp. sequence obtained from a Noctilio albiventris (KP715475) from French Guiana. The present study provided valuable insights into the diversity of Bartonella genotypes infecting bats from two ecoregions (Amazonia and Cerrado) in midwestern Brazil and emphasized that further studies should be conducted regarding the description and evaluation of different lineages of Bartonella in wild small mammals and their ectoparasites in different Brazilian biomes.

A rapid field-ready electrical biosensor consisting of bismuthine-derived Au island decorated BiOCl nanosheets for Raphidiopsis raciborskii detection in freshwater.

Cyanobacteria play an essential role in nutrient cycling in aquatic ecosystems. However, certain species adversely affect the environment and human health by causing harmful cyanobacterial algal blooms (cyanoHABs) and producing cyanotoxins. To address this issue, continuous cyanoHAB monitoring has been considered; however, a gold standard has not yet been established. In this study, we aimed to develop a dual DNA-targeting capacitive-type biosensor for rapid field-ready monitoring of Raphidiopsis raciborskii, a causative species of cyanoHAB. To enhance the sensing signal, a plate-like Au-BiOCl nanocomposite was synthesized using a spontaneous carbonation process without additional additives. The alternating-current electrothermal flow (ACEF) technique was applied to enable rapid DNA and probe binding within 10 min. The limits of detection (LODs) for R. raciborskii RubisCO large subunit (rbcL) and RNA polymerase beta subunit (rpoB) genes diluted in deionized (DI) water were 4.89 × 10-17 and 3.89 × 10-17 M, respectively. Furthermore, the LODs of R. raciborskii rbcl and rpoB diluted in freshwater containing HAB were 2.55 × 10-16 and 3.84 × 10-16 M, respectively, demonstrating the field-ready applicability of the device. The fabricated cyanobacterial DNA-sensing platform enabled powerful species-specific detection using a small sample volume and low target concentration without a nucleic acid amplification step, dramatically reducing the detection time. This study has considerable implications for detecting HABs, early warning systems, and species-specific environmental monitoring technology.

Biological Control of White Rot in Apple Using Bacillus spp.

Apple white rot, caused by <i>Botryosphaeria dothidea</i>, is one of the important diseases in Korea. <i>B. dothidea</i> can cause pre- and postharvest decay on apple fruit as well as canker and dieback of apple trees. In this study, we isolated bacteria from the trunk of apple trees and tested their antagonistic activity against <i>B. dothidea</i>. Five bacterial isolates (23-168, 23-169, 23-170, 23-172, and 23-173) were selected that were most effective at inhibiting the mycelial growth of the pathogens. The isolate 23-172 was identified as <i>Bacillus amyloliquefaciens</i> and four isolates 23-168, 23-169, 23-170, and 23-173 were identified as <i>Bacillus velezensis</i> by RNA polymerase beta subunit (<i>rpoB</i>) and DNA gyraseA subunit (<i>gyrA</i>) gene sequencing. All isolates showed strong antagonistic activity against <i>B. dothidea</i> as well as <i>Colletotrichum fructicola</i> and <i>Diaporthe eres</i>. All isolates exhibited cellulolytic, proteolytic and phosphate solubilizing activities. In particular, two isolates 23-168, 23-169 were shown to significantly reduce the size of white rot lesions in pretreated apple fruits. These results will provide the basis for the development of a fungicide alternative for the control of white rot of apple.

Tracing the Evolutionary Pathways of Serogroup O78 Avian Pathogenic Escherichia coli.

Avian pathogenic E. coli (APEC) causes severe economic losses in the poultry industry, and O78 serogroup APEC strains are prevalent in chickens. In this study, we aimed to understand the evolutionary pathways and relationships between O78 APEC and other E. coli strains. To trace these evolutionary pathways, we classified 3101 E. coli strains into 306 subgenotypes according to the numbers and types of single nucleotide polymorphisms (RST0 to RST63-1) relative to the consensus sequence (RST0) of the RNA polymerase beta subunit gene and performed network analysis. The E. coli strains showed four apparently different evolutionary pathways (I-1, I-2, I-3, and II). The thirty-two Korean O78 APEC strains tested in this study were classified into RST4-4 (45.2%), RST3-1 (32.3%), RST21-1 (12.9%), RST4-5 (3.2%), RST5-1 (3.2%), and RST12-6 (3.2%), and all RSTs except RST21-1 (I-2) may have evolved through the same evolutionary pathway (I-1). A comparative genomic study revealed the highest relatedness between O78 strains of the same RST in terms of genome sequence coverage/identity and the spacer sequences of CRISPRs. The early-appearing RST3-1 and RST4-4 prevalence among O78 APEC strains may reflect the early settlement of O78 E. coli in chickens, after which these bacteria accumulated virulence and antibiotic resistance genes to become APEC strains. The zoonotic risk of the conventional O78 APEC strains is low at present, but the appearance of genetically distinct and multiple virulence gene-bearing RST21-1 O78 APEC strains may alert us to a need to evaluate their virulence in chickens as well as their zoonotic risk.

Pilot study on the distribution of caseous lymphadenitis in Korean native goats and the relationship between sex and age in disease occurrence

Caseous lymphadenitis (CLA) caused by Corynebacterium pseudotuberculosis is a chronic disease that affects goats. The Korean native goat (KNG) is the most popular goat breed raised in Korea. In this study, the distribution of CLA in the KNG population and the relevance of sex and age in disease development were determined. From March 2020 to February 2021, 1,177 KNGs from 110 farms were assessed. The distribution of CLA in animals was determined by a veterinary inspector who performed gross examinations of goat carcasses and confirmed diagnoses via polymerase chain reaction and bacteriological examination. The CLA detection rate in the KNG population was 19.80%, and more than half of the farms were affected by the disease (56.36%). A statistically significant difference was observed among the sex groups, with castrated males (13.98%) having the lowest detection rate, followed by intact males (22.48%) and females (24.09%), suggesting that castration has a positive effect on reducing the risk of CLA (p < 0.05). In terms of age groups, an increased detection rate of 28.16% was observed in the late adult (>2 years) group. Phylogenetic analysis indicated that the RNA polymerase beta subunit-encoding gene can effectively differentiate biovar ovis from biovar equi and can be used for further epidemiological studies of C. pseudotuberculosis. This is the first nationwide surveillance study of CLA distribution to confirm the continuous occurrence of CLA in Korean goat farms. Future studies should include risk factor analyses of CLA based on herd levels to prevent and control this disease in Korea.

Biomarker metaproteomics for relative taxa abundances across soil organisms

Soil organisms are often classified using methods targeting individual groups of taxa (e.g., bacteria, fungi and invertebrates), which hampers our ability to directly compare the relative abundance of different groups across environmental gradients. We posit that the use of protein biomarkers could help to provide a more real representation of the cross-kingdom soil microbial populations. Here, we tested if the abundant proteins ATP synthase F(0) complex (ATPS), elongation factors (EF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), GroEL, pyruvate dehydrogenase (PyrDH), RNA polymerase beta chain (RNAP), and translation initiation factor 2 (TIF) could be used to describe the taxonomic composition of microbial communities. As positive control, we used a mock community with different relative abundances of algae, archaea, bacteria, and viruses. We tested this approach on a previously published soil metaproteomes from which we randomly selected samples from forests, grasslands, and shrublands (each n = 10). Unfortunately, the biomarker approach is not feasible for viruses as these organisms do not share single genes. All biomarkers showed decent accuracy to determine the relative abundances of archaea, bacteria, and eukaryota in the mock community. However, false positive hits dominated on phylum level probably due to sequence homology. Archaeal proteins were only detected in the soil samples when EF was used as biomarker at an abundance of 0.7%. Bacteria dominated the EF-metaproteome and were most abundant in shrublands (64.4%) while eukaryotes were more abundant in forests (25.6%). In compliance with previously published results, the correlation analysis revealed the impact of mean annual temperature and pH on both bacteria and eukaryota. Our approach not only shows the potential to use biomarker metaproteomics to unveil the relative taxa abundances across soil organisms but also the need to create mock communities comprising members of all soil taxa.

Mycoplasma sp. Detection in the State Endangered Illinois Alligator Snapping Turtle (Macrochelys temminckii)

Abstract The alligator snapping turtle (Macrochelys temminckii) is the largest freshwater turtle species in North America and is classified as state endangered in Illinois. Head start programs that include health assessments and pathogen detection are being conducted to restore this species to its historic range. Physical examinations and oral and cloacal swabs were collected from 97 head start alligator snapping turtles prerelease and from 58 recaptured turtles postrelease in 2014. Conventional polymerase chain reaction and sequencing targeting the RNA polymerase beta subunit gene and the 16S-23S ribosomal RNA intergenic spacer detected a single prerelease individual with Mycoplasma sp. with closest relation to Mycoplasma spp. of other freshwater turtles. This individual did not display any clinical signs at the time of release or sampling. Mycoplasmosis has been characterized as a disease of conservation concern in tortoises and may represent a threat to small and fragmented chelonian populations. Findings of this study indicate that Mycoplasma sp. DNA is present in an Illinois alligator snapping turtle, and future investigation into the impact of this disease may support conservation efforts.

Fructobacillus papyriferae sp. nov., Fructobacillus papyrifericola sp. nov., Fructobacillus broussonetiae sp. nov. and Fructobacillus parabroussonetiae sp. nov., isolated from paper mulberry in Taiwan.

Five Gram-stain-positive strains (M1-10T, M1-13, M1-21T, M2-14T and S1-1T) were isolated from paper mulberry (Broussonetia papyrifera) in Taiwan. Cells were rod-shaped, non-motile, non-haemolytic, asporogenous, facultatively anaerobic, heterofermentative, and did not exhibit catalase and oxidase activities. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these novel strains belonged to the genus Fructobacillus. On the basis of 16S rRNA gene sequence similarities, the type strains of Fructobacillus fructosus and Fructobacillus durionis were the closest neighbours to strains M1-10T, M1-13, M1-21T, M2-14T and S1-1T. Sequence analyses of concatenated two partial housekeeping genes, the RNA polymerase beta subunit (rpoC) and recombinase A (recA) also indicated that the novel strains belonged to the genus Fructobacillus. The 16S rRNA and concatenated rpoC and recA gene sequence similarities between strains M1-10T and M1-13 were 100 %, respectively. The average nucleotide identity values of M1-10T, M1-21T, M2-14T and S1-1T with F. fructosus and F. durionis were 75.1-78.9% and 76.5-77.5 %, respectively. The digital DNA-DNA hybridization values were 19.7-21.5% and 19.6-20.4 %, respectively. Phenotypic and genotypic test results demonstrated that these strains represent four novel species of the genus Fructobacillus, for which the names Fructobacillus papyriferae sp. nov., Fructobacillus papyrifericola sp. nov., Fructobacillus broussonetiae sp. nov. and Fructobacillus parabroussonetiae sp. nov. are proposed with the type strains M1-10T (=BCRC 81237T=NBRC 114433T), M1-21T (=BCRC 81239T=NBRC 114435T), M2-14T (=BCRC 81240T=NBRC 114436T) and S1-1T (=BCRC 81241T=NBRC 114437T), respectively.

Molecular identification of streptomycetes by exploiting RNA polymerase beta-subunit (rpoB) gene in Saudi arabia

This study was designed with the aim of exploring the efficacy of RNA polymerase beta subunit, rpoB gene analysis for the identification of streptomycetes. A characteristic marine environment distinguished by their content of streptomycetes were targeted, and two organisms of the genus Streptomyces were isolated in Saudi Arabia. The two Streptomyces spp., named isolates EH1 and EH2 were isolated and subjected to the regular phenotypic characterization including morphological and physiological studies, analyses of cells hydrolysates, detecting their antibacterial activities and pocks formation ability. These isolates showed inhibitory activity of gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. The two isolates gave positive results in pocks formation test, which indicated that both must belong to the genus Streptomyces. Molecular identification of both isolates was accomplished through analysis of the beta-subunit of the RNA polymerase gene (rpoB) to assist in species identification. The target fragments 352 bp were amplified and detected in both isolates using agarose gel electrophoresis, indicating that they belong to the genus Streptomyces. The amplified product of the isolate EH1 was sequenced and 296 bp continuous sequence was determined (GenBank accession MK569762.1). By matching the obtained sequence with the Streptomyces DNA sequence databases, the isolate EH1 was found very similar to Streptomyces labedae. The study concluded with the possibility of using molecular analysis of RNA polymerase beta subunit gene to correctly identify streptomycetes to the species level.

Beyond RuBisCO: convergent molecular evolution of multiple chloroplast genes in C4 plants.

BackgroundThe recurrent evolution of the C4 photosynthetic pathway in angiosperms represents one of the most extraordinary examples of convergent evolution of a complex trait. Comparative genomic analyses have unveiled some of the molecular changes associated with the C4 pathway. For instance, several key enzymes involved in the transition from C3 to C4 photosynthesis have been found to share convergent amino acid replacements along C4 lineages. However, the extent of convergent replacements potentially associated with the emergence of C4 plants remains to be fully assessed. Here, we conducted an organelle-wide analysis to determine if convergent evolution occurred in multiple chloroplast proteins beside the well-known case of the large RuBisCO subunit encoded by the chloroplast gene rbcL.MethodsOur study was based on the comparative analysis of 43 C4 and 21 C3 grass species belonging to the PACMAD clade, a focal taxonomic group in many investigations of C4 evolution. We first used protein sequences of 67 orthologous chloroplast genes to build an accurate phylogeny of these species. Then, we inferred amino acid replacements along 13 C4 lineages and 9 C3 lineages using reconstructed protein sequences of their reference branches, corresponding to the branches containing the most recent common ancestors of C4-only clades and C3-only clades. Pairwise comparisons between reference branches allowed us to identify both convergent and non-convergent amino acid replacements between C4:C4, C3:C3 and C3:C4 lineages.ResultsThe reconstructed phylogenetic tree of 64 PACMAD grasses was characterized by strong supports in all nodes used for analyses of convergence. We identified 217 convergent replacements and 201 non-convergent replacements in 45/67 chloroplast proteins in both C4 and C3 reference branches. C4:C4 branches showed higher levels of convergent replacements than C3:C3 and C3:C4 branches. Furthermore, we found that more proteins shared unique convergent replacements in C4 lineages, with both RbcL and RpoC1 (the RNA polymerase beta’ subunit 1) showing a significantly higher convergent/non-convergent replacements ratio in C4 branches. Notably, more C4:C4 reference branches showed higher numbers of convergent vs. non-convergent replacements than C3:C3 and C3:C4 branches. Our results suggest that, in the PACMAD clade, C4 grasses experienced higher levels of molecular convergence than C3 species across multiple chloroplast genes. These findings have important implications for our understanding of the evolution of the C4 photosynthesis pathway.

Infection of the Asian gray shrew Crocidura attenuata (Insectivora: Soricidae) with Sarcocystis attenuati n. sp. (Apicomplexa: Sarcocystidae) in China

BackgroundData on the genus Sarcocystis in insectivores are limited. The Asian gray shrew Crocidura attenuata is one of the most common species of the insectivore family Soricidae in South Asia and Southeast Asia. To our knowledge, species of Sarcocystis have never been recorded previously in this host.MethodsTissues were obtained from 42 Asian gray shrews caught in 2017 and 2018 in China. Sarcocysts were observed using light microscopy (LM) and transmission electron microscopy (TEM). To describe the parasite life cycle, muscle tissues of the host infected with sarcocysts were force-fed to two beauty rat snakes Elaphe taeniura. Individual sarcocysts from different Asian gray shrews, and oocysts/sporocysts isolated from the small intestines and feces of the experimental snakes, were selected for DNA extraction, and seven genetic markers, namely, two nuclear loci [18S ribosomal DNA (18S rDNA) and internal transcribed spacer region 1 (ITS1)], three mitochondrial genes [cytochrome oxidase subunit 1 (cox1), cox3 and cytochrome b], and two apicoplast genes (RNA polymerase beta subunit and caseinolytic protease C), were amplified, sequenced and analyzed.ResultsSarcocysts were found in 17 of the 42 (40.5%) Asian gray shrews. Under LM, the microscopic sarcocysts showed saw- or tooth-like protrusions measuring 3.3–4.5 μm. Ultrastructurally, the sarcocyst wall contained numerous lancet- or leaf-like villous protrusions, similar to those described for type 9h of the common cyst wall classification. The experimental beauty rat snakes shed oocysts/sporocysts measuring 11.9–16.7 × 9.2–10.6 μm with a prepatent period of 10–11 days. Comparison of the newly obtained sequences with those previously deposited in GenBank revealed that those of 18S rDNA and cox1 were most similar to those of Sarcocystis scandentiborneensis recorded in the tree shrews Tupaia minor and Tupaiatana (i.e., 97.6–98.3% and 100% identity, respectively). Phylogenetic analysis based on 18S rDNA or ITS1 sequences placed this parasite close to Sarcocystis spp. that utilize small animals as intermediate hosts and snakes as the known or presumed definitive host. On the basis of morphological and molecular characteristics and host specificity, the parasite was proposed as a new species, named Sarcocystis attenuati.ConclusionsSarcocysts were recorded in Asian gray shrews, to our knowledge for the first time. Based on morphological and molecular characterization, a new species of parasite is proposed: Sarcocystisattenuati. According to the LM and TEM results, S. attenuati sarcocysts are distinct from those of Sarcocystis spp. in other insectivores and those of S. scandentiborneensis in tree shrews. The 18S rDNA or cox1 sequences of Sarcocystis attenuati shared high similarity with those of Sarcocystisscandentiborneensis, Sarcocystis zuoi, Sarcocystis cf. zuoi in the Malayan field rat, and Sarcocystis sp. in the greater white-toothed shrew. Therefore, we suggest that more research on the relationships of these closely related taxa should be undertaken in the future.Graphical abstract

Rid7C, a Member of the YjgF/YER057c/UK114 (Rid) Protein Family, Is a Novel Endoribonuclease That Regulates the Expression of a Specialist RNA Polymerase Involved in Differentiation in Nonomuraea gerenzanensis.

The YjgF/YER057c/UK114 (Rid) is a protein family breadth conserved in all domains of life and includes the widely distributed archetypal RidA (YjgF) subfamily and seven other subfamilies (Rid1 to Rid7). Among these subfamilies, RidA is the only family to have been biochemically well characterized and is involved in the deamination of the reactive enamine/imine intermediates. In this study, we have characterized a protein of the Rid7 subfamily, named Rid7C, in Nonomuraea gerenzanensis, an actinomycete that is characterized by the presence of two types of RNA polymerases. This is due to the coexistence in its genome of two RNA polymerase (RNAP) β chain-encoding genes, rpoB(S) (the wild-type rpoB gene) and rpoB(R) (a specialist, mutant-type rpoB gene) that controls A40926 antibiotic production and a wide range of metabolic adaptive behaviors. Here, we found that expression of rpoB(R) is regulated posttranscriptionally by RNA processing in the 5' untranslated region (UTR) of rpoB(R) mRNA and that the endoribonuclease activity of Rid7C is responsible for mRNA processing, thereby overseeing several tracts of morphological and biochemical differentiation. We also provide evidence that Rid7C may be associated with RNase P M1 RNA, although M1 RNA is not required for rpoB(R) mRNA processing in vitro, and that Rid7C endoribonuclease activity is inhibited by A40926, suggesting the existence of a negative feedback loop in A40926 production and a role of the endogenous synthesis of A40926 in the modulation of biochemical differentiation in this microorganism. IMPORTANCE The YjgF/YER057c/UK114 family includes many proteins with diverse functions involved in detoxification, RNA maturation, and control of mRNA translation. We found that Rid7C is an endoribonuclease that is involved in processing of rpoB(R) mRNA, coding for a specialized RNA polymerase beta subunit that oversees morphological differentiation and A40926 antibiotic production in Nonomuraea gerenzanensis. Rid7C-mediated processing promotes rpoB(R) mRNA translation and antibiotic production, while Rid7C endoribonuclease activity is inhibited by A40926, suggesting a role of the endogenous synthesis of A40926 in modulation of biochemical differentiation in this microorganism. Finally, we show that recombinant Rid7C copurified with M1 RNA (the RNA subunit of RNase P) from Escherichia coli extract, suggesting a functional interaction between Rid7C and M1 RNA activities.

Comparative genomics of bovine mastitis-origin Staphylococcus aureus strains classified into prevalent human genotypes

Humans may serve as a reservoir host of Staphylococcus aureus, resulting in transmission to animals. Previously, we used RNA polymerase beta subunit gene (rpoB)-based genotyping and classified S. aureus strains into rpoB sequence types (RSTs). According to our previous work, the predominant genotypes of S. aureus in humans and cows differ in Korea, but some predominant genotypes (RST4–1 and RST2–1) in humans have been isolated from bovine mastitis. Therefore, it needs to be determined whether some strains of the predominant human genotypes have adapted to or caused occasional infections in cows. We determined the whole genome sequences of 2 bovine mastitis-origin strains, PMB179 (RST4–1) and PMB196 (RST2–1), and performed comparative genomics with the corresponding RST4–1 and RST2–1 S. aureus strains in the NCBI database. We identified 257 and 180 pseudogenes among 131 RST4–1 and 54 RST2–1 strains, respectively, for the comparison of pseudogene profiles. RST4–1 strains shared more common pseudogenes than RST2–1 strains, and some epidemiologically related strains shared common pseudogenes. However, most of the pseudogenes were strain-specific, and diverse pseudogene profiles were apparent in both the RST4–1 and RST2–1 strains. Furthermore, analysis of the mobile genetic elements, virulence genes, and antibiotic resistance genes revealed no molecular markers to differentiate PMB179 and PMB196 from human strains. Interestingly, the collective comparison of RST4–1 or RST2–1 strains revealed cumulative acquisition steps of genomic islands and antibiotic resistance genes. In conclusion, our data support PMB179 and PMB196 causing occasional infections that result in bovine mastitis.

Investigation of Mutations in the Rifampin-Resistance-Determining Region of the rpoB Gene of Brucella melitensis by Gene Analysis

Background: RNA polymerase beta subunit (rpoB) gene analysis in bacterial communities is known as a method for determining rifampin sensitivity and genetic diversity among Brucella spp. Detection of antibiotic resistance among Brucella isolates can be a critical approach to control brucellosis. However, rpoB gene analysis of Brucella melitensis for assessing rifampicin resistance has not yet been performed in Iran, which is considered an endemic area for brucellosis. Objectives: The aim of this study was to analyze the whole sequence of rpoB genes of different B. melitensis isolates from humans to identify the single-nucleotide polymorphisms (SNPs) and mutations related to rifampin resistance and to analyze the genetic diversity of these bacteria in Iran. Methods: Between 2017 and 2019, a total of 156 blood samples along with 12 synovial fluid specimens were collected from brucellosis patients in different Iranian provinces and subjected to bacterial culture in Brucella selective media. Brucella identification was carried out using classical biotyping and molecular examinations. Polymerase chain reaction (PCR)-based amplification of the rpoB gene was performed by specific rpoB primers for whole gene sequencing. The antimicrobial susceptibility of Brucella isolates was assessed using disk diffusion susceptibility tests and minimal inhibitory concentration (MIC) methods. The presence of rifampin-binding sites and SNPs were investigated through rpoB whole gene sequencing. Results: Clinical B. melitensis isolates were obtained from blood (13) and synovial fluid (1) samples of patients from different regions of Iran. The results of MIC and disk diffusion susceptibility tests showed that all the isolates were sensitive to rifampin except for one isolate showing intermediate rifampin resistance based on the standards defined for slow-growing bacteria by the Clinical and Laboratory Standards Institute (CLSI). Gene analysis for identifying the mutations related to rifampin resistance and investigating genetic diversity showed that none of the B. melitensis isolates had missense mutations, confirming the susceptibility of all the studied isolates to rifampin. Conclusions: The present study revealed that rpoB gene analysis could be used for the efficient and precise identifying of the mutations related to rifampin resistance, investigating rifampin binding sites, and genotyping Brucella species. Furthermore, the identification of B. melitensis isolates with intermediate resistance to rifampicin highlighted the importance of periodically carrying out antibiotic susceptibility testing. The molecular detection of rpoB mutations in different Brucella isolates may help to prevent the spread of rifampin-resistant Brucella spp. among humans and livestock.

A database of sequenced genomes of different Streptomyces albus J1074 strains and uses thereof

Streptomyces albus J1074 has recently emerged as a powerful chassis strain for natural product research and discovery, as well as model to investigate various aspects of actinobacterial biology. A number of genetic tools have been developed to facilitate the use of this strain for the aforementioned purposes. One of the promising approaches is to introduce into J1074 genome mutations that would improve the antibiotic-producing capacity of J1074. Particularly, we reported a collection of spontaneous and genomically engineered J1074 mutants carrying mutation in genes for ribosomal protein S12, RNA polymerase beta subunit etc. We took advantage of this collection to build an in-house database which would host (both current and future) the primary and curated sequencing data for these mutants. The database is available at https://biotools.online/media/. The main benefits of the database lie in the known pedigree of the strains, which allows deep interpretation of the data. For example, there is lively – and unresolved – debate on the origins and consequences of the GC composition in actinobacteria. A better understanding of this issue should improve our knowledge of genome evolution in bacteria and will have a number of biotechnological ramifications. We used our Streptomyces albus J1074 dataset as an experimental model to reveal genome-wide spectrum of mutation, which appears to be biased towards elevated GC content. We also included the high-quality genomes of the other streptomycetes into our databse for comparative purposes. The genomic GC content in streptomycetes varies from 75 % to 66.5 %, with median value being 72 %. The GC content of secondary metabolic genes of S. coelicolor is less variable than that of primary metabolic genes, an indicative of different selection pressure on these gene groups. Along with selective constraints, the peculiarities of Streptomyces DNA mismatch repair might contribute to the skewed GC content of their genomes. Further uses of the database may include the development of a more precise knowledge of the mutation rate as well as population genetic processes within this species and genus.

Identification and validation of reference genes for reliable analysis of differential gene expression during antibiotic induced persister formation in Klebsiella pneumoniae using qPCR

The study of differential gene expression in persister cells is compounded by ceasure of conventional cellular metabolic pathways during persistence. There is, hence, a requirement to identify and validate suitable reference genes whose expression remains stable during persistence. We evaluated the suitability of five genes viz. dnaJ, groEL, rpoB, kp751, kp4432 as references to study gene expression using real-time polymerase chain reaction (qPCR) during persister cell formation in Klebsiella pneumoniae. Results obtained showed that while dnaJ and groEL suffered from unstable expression; rpoB, kp751 and kp4432 showed stable expression. Further, it was observed that data normalization using either of the stable genes viz. rpoB, kp751, kp4432 alone, resulted in either too low expression levels or too high variation among replicates. Our study indicates the concurrent use of kp4432 and rpoB as reference genes to be the most suitable for reliable analysis of differential gene expression during antibiotic induced persister formation in K. pneumoniae. kp4432 and rpoB encode NAD-dependant phenylacetaldehyde dehydrogenase and DNA-directed RNA polymerase beta subunit respectively. The outcome of this study will increase the utility of qPCR in studying the temporal changes in gene expression during persistence. The study will also aid in understanding mechanisms underlying persister cell formation particularly in K. pneumoniae.

Genetic diversity of Bartonella species in small mammals in the Qaidam Basin, western China

Investigation of the prevalence and diversity of Bartonella infections in small mammals in the Qaidam Basin, western China, could provide a scientific basis for the control and prevention of Bartonella infections in humans. Accordingly, in this study, small mammals were captured using snap traps in Wulan County and Ge’ermu City, Qaidam Basin, China. Spleen and brain tissues were collected and cultured to isolate Bartonella strains. The suspected positive colonies were detected with polymerase chain reaction amplification and sequencing of gltA, ftsZ, RNA polymerase beta subunit (rpoB) and ribC genes. Among 101 small mammals, 39 were positive for Bartonella, with the infection rate of 38.61%. The infection rate in different tissues (spleens and brains) (χ2 = 0.112, P = 0.738) and gender (χ2 = 1.927, P = 0.165) of small mammals did not have statistical difference, but that in different habitats had statistical difference (χ2 = 10.361, P = 0.016). Through genetic evolution analysis, 40 Bartonella strains were identified (two different Bartonella species were detected in one small mammal), including B. grahamii (30), B. jaculi (3), B. krasnovii (3) and Candidatus B. gerbillinarum (4), which showed rodent-specific characteristics. B. grahamii was the dominant epidemic strain (accounted for 75.0%). Furthermore, phylogenetic analysis showed that B. grahamii in the Qaidam Basin, might be close to the strains isolated from Japan and China. Overall, we observed a high prevalence of Bartonella infection in small mammals in the Qaidam Basin. B. grahamii may cause human disease, and the pathogenicity of the others Bartonella species needs further study, the corresponding prevention and control measures should be taken into consideration.

Lincomycin-Induced Secondary Metabolism in Streptomyces lividans 66 with a Mutation in the Gene Encoding the RNA Polymerase Beta Subunit.

Activating the genetic potential of Streptomyces strains to produce secondary metabolites can improve the production of useful biologically active compounds and facilitate the discovery of novel biologically active compounds. In this study, we found that Streptomyces lividans carrying the R440H mutation in rpoB, encoding the RNA polymerase beta subunit, grown in the presence of lincomycin at concentrations below the minimum inhibitory concentration (MIC) produced abundant amounts of actinorhodin and certain cryptic secondary metabolites despite culture conditions that restrict their production by the wild-type strain. The results indicate that lincomycin at concentrations below the MIC may strongly potentiate secondary metabolite production by Streptomyces strains carrying a specific rpoB mutation. In this study, we report an interesting phenomenon induced by combining the positive effects of certain rpoB mutations and concentration-dependent responses to lincomycin on secondary metabolism in S. lividans 66 and discuss the mechanisms and their applicability in exploring cryptic secondary metabolite production in streptomycetes.