Beta Subunit Of RNA Polymerase Research Articles

Isolation and evaluation of Bacillus subtilis RSS-1 as a potential biocontrol agent against Sclerotinia sclerotiorum on oilseed rape

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases of oilseed rape in the world. Because of the absence of resistant varieties and the disadvantages of chemical control, application of antifungal microbes has become an eco-friendly and effective measure to control this disease. In this study, Bacillus subtilis strain RSS-1, isolated from soil samples, was identified based on morphological, physiological and biochemical tests, and DNA gyrase subunit A (gyrA), gyrB, DNA-directed RNA polymerase subunit beta (rpoB) and rpoC gene sequence analysis. It significantly inhibited mycelial growth and sclerotial production of S. sclerotiorum in vitro. In greenhouse experiments, all three tested concentrations (106, 107, 108 cfu mL−1) of cell fermentation broth and culture filtrate significantly reduced the severity of sclerotinia stem rot on oilseed rape (P < 0.05). RSS-1 was more effective at reducing disease severity when applied 24 h before inoculation with S. sclerotiorum than at 24 h post inoculation, suggesting that RSS-1 should be applied as a prophylactic rather than a curative biological agent. Colonization tests indicated that the population density of RSS-1 on rapeseed leaves significantly decreased (P < 0.05) over 6 days. However, RSS-1 could stably colonize in rhizospheric soil of rapeseed over 30 days. Challenge inoculation tests showed that RSS-1 significantly inhibited the activities of polygalacturonase and cellulase and accumulation of oxalic acid during the S. sclerotiorum infection. These results suggest that RSS-1 was a potential biological agent for controlling sclerotinia stem rot caused by S. sclerotiorum on oilseed rape.

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.

Trends in the incidence of Rifampicin resistant Mycobacterium tuberculosis infection in northeastern Nigeria

Mycobacterium tuberculosis is a respiratory bacterial pathogen that causes Tuberculosis. About one-quarter of the global population is infected, most of which are asymptomatic with a 5–15% risk of developing active disease across their lifespan. The progression of the disease has been enhanced by HIV coinfection and the emergence of Mycobacterium tuberculosis strains that are resistant to first-line anti-TB drugs, especially Rifampicin. Rifampicin resistance is brought about by mutation in the gene coding for beta-subunit of the enzyme RNA polymerase (rpoB) and is a surrogate marker for multidrug-resistant Tuberculosis (MDR-TB). Herein, using the GeneXpert MTB/RIF molecular technique, we examined the incidence of Mycobacterium tuberculosis infection and evaluate rpoB gene mutation among Mycobacterium tuberculosis isolates identified from presumptive pulmonary tuberculosis (PTB) and extra-pulmonary tuberculosis (EPTB) patients attending the directly observed treatment short-course (DOTS) clinic, University of Maiduguri Teaching Hospital, Nigeria. A total of two thousand four hundred and fifty-one (2451) presumptive TB cases (PTB = 2279, EPTB = 172) were analyzed from January 2018 to July 2019. The incidence of Mycobacterium tuberculosis infection was 22.68% (Mean±S.E = 46.33 ± 15.41). The incidence of Rifampicin-resistant Mycobacterium tuberculosis was 4.50% (Mean±S.E = 2.08 ± 1.02). The incidence of Mycobacterium tuberculosis and HIV co-infection was 16.59%, while the incidence of Rifampicin-resistant Mycobacterium tuberculosis and HIV co-infection was 4.23%. The peak incidence of M. tuberculosis infection was observed during the first quarter of each year and this variation was observed to be statistically significant (f = 9.34606; p < .05; CI95; p = 0.000068). Incidence of Rifampicin-resistant Mycobacterium tuberculosis was observed to be more prevalent among female patients that are 15 years or older (Fischer's exact test = 0.533; p < .05; CI95; p = 0.465348). The incidence rates reported herein are higher than the WHO-reported national average. By this report, it can be affirmed that the incidence of Mycobacterium tuberculosis infection in northeastern Nigeria is increasing and the rate of infection follows a seasonal trend.

Andrographis Echioides Molecular Characterization and Phylogenetic Study Using DNA Barcoding Technique

With the advancement in various molecular diagnostic tools, DNA Barcoding has emerged as a gold standard molecular diagnostic tool across the globe. Since ancient times, medicinal plants have been widely used in Indian Ayurvedic medicine for treating a variety of ailments. Plants of the genus Andrographis have been extensively used for treating different types of ailments. In this study, rarely studied medicinal plant species were isolated, sequenced at the genetic level and studied for their evolutionary characteristics using phylogenetic analysis. In the present study, the identity of A. echioides was confirmed by targeting different barcoding genes such as ribulose-bisphosphate carboxylase, internal transcribed spacer, RNA polymerase-beta subunit, maturase K, and photosystem II protein D1 genes using a phylogenetic approach. After successful isolation and amplification of genomic DNA, specific primers were utilised for sequencing of each barcoding gene, followed by nucleotide BLAST analysis to determine the sequence percent identity of each gene with that from other plant species. The best homologs were then utilised for conducting phylogenetic analysis which confirmed the identity of the plant as Andrographis echioides.

Mycobacterium senriense sp. nov., a slowly growing, non-scotochromogenic species, isolated from sputum of an elderly man.

A slowly growing mycobacteria, identified as strain TY59T, was isolated from sputum of an elderly man with pneumonia. Sequencing of the 16S rRNA gene indicated that this strain was similar to members of the Mycobacterium avium complex and closely related species. Strain TY59T has highest 16S rRNA gene sequence similarities to the type strains of Mycobacterium colombiense (99.80 % sequence similarity), Mycobacterium vulneris (99.74 %), Mycobacterium timonense (99.54 %), Mycobacterium avium subsp. avium (99.54 %) and Mycobacterium avium subsp. silvaticum (99.54 %). Analysis of the internal transcribed spacer (ITS) and DNA-directed RNA polymerase subunit beta (rpoB) sequences gave similar results to the 16S rRNA gene analysis. The closest species to strain TY59T were M. colombiense and M. vulneris with 97.90-98.25 % identity in ITS and 96.4-96.6 % in rpoB. The strain's 65 kDa heat shock protein (hsp65) gene was different from those of M. vulneris, M. colombiense and M. avium subsp. silvaticum with 72.4-74.2 % identity. Average nucleotide identity results showed a 93.4 % match to M. vulneris as the maximum value. Phenotypically, the non-chromogenicity, rough colonies, growth at 42 °C, negative results for nitrate reduction, β-glucosidase and Tween 80 hydrolysis, and positive results for catalase activity set this strain apart from closely related species. We propose that Mycobacterium senriense sp. nov. is a novel species of slowly growing mycobacteria. The type strain is TY59T (RIMD 1371001T=CIP 111917T).

Mycobacterium tuberculosis Affects Protein and Lipid Content of Circulating Exosomes in Infected Patients Depending on Tuberculosis Disease State.

Tuberculosis (TB), which is caused by the bacterium Mycobacterium tuberculosis (Mtb), is still one of the deadliest infectious diseases. Understanding how the host and pathogen interact in active TB will have a significant impact on global TB control efforts. Exosomes are increasingly recognized as a means of cell-to-cell contact and exchange of soluble mediators. In the case of TB, exosomes are released from the bacillus and infected cells. In the present study, a comprehensive lipidomics and proteomics analysis of size exclusion chromatography-isolated plasma-derived exosomes from patients with TB lymphadenitis (TBL) and treated as well as untreated pulmonary TB (PTB) was performed to elucidate the possibility to utilize exosomes in diagnostics and knowledge building. According to our findings, exosome-derived lipids and proteins originate from both the host and Mtb in the plasma of active TB patients. Exosomes from all patients are mostly composed of sphingomyelins (SM), phosphatidylcholines, phosphatidylinositols, free fatty acids, triacylglycerols (TAG), and cholesterylesters. Relative proportions of, e.g., SMs and TAGs, vary depending on the disease or treatment state and could be linked to Mtb pathogenesis and dormancy. We identified three proteins of Mtb origin: DNA-directed RNA polymerase subunit beta (RpoC), Diacyglycerol O-acyltransferase (Rv2285), and Formate hydrogenase (HycE), the latter of which was discovered to be differently expressed in TBL patients. Furthermore, we discovered that Mtb infection alters the host protein composition of circulating exosomes, significantly affecting a total of 37 proteins. All TB patients had low levels of apolipoproteins, as well as the antibacterial proteins cathelicidin, Scavenger Receptor Cysteine Rich Family Member (SSC5D), and Ficolin 3 (FCN3). When compared to healthy controls, the protein profiles of PTB and TBL were substantially linked, with 14 proteins being co-regulated. However, adhesion proteins (integrins, Intercellular adhesion molecule 2 (ICAM2), CD151, Proteoglycan 4 (PRG4)) were shown to be more prevalent in PTB patients, while immunoglobulins, Complement component 1r (C1R), and Glutamate receptor-interacting protein 1 (GRIP1) were found to be more abundant in TBL patients, respectively. This study could confirm findings from previous reports and uncover novel molecular profiles not previously in focus of TB research. However, we applied a minimally invasive sampling and analysis of circulating exosomes in TB patients. Based on the findings given here, future studies into host–pathogen interactions could pave the way for the development of new vaccines and therapies.

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.

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

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

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

Proteome wide screening for identification of putative drug target gene in Nautella italica and structure-based ligand screening for therapeutic candidates

In silico based subtractive genomic approaches were employed to identify the key drug targets for an opportunistic pathogen Nautella italica, a member of the marine Roseobacter clade that causes bleaching disease in the temperate-marine red macro algae, Delisea pulchra. The aim of this study is to propose new active ligands against bleaching disease seen in algae. Using comparative and subtractive genomic approach, a set of 21 proteins were identified as the therapeutic drug target proteins for algal bleaching. This core set of drug targets has been analyzed for network topology using string network analysis and major hub gene identified by CytoHubba was rpoB (DNA directed RNA Polymerase subunit beta). The three-dimensional structure of rpoB was built by comparative modelling and used to perform a virtual screening of Zinc database by DOCK Blaster server. The 50 top scored compounds were screened for toxicity analysis by OSIRIS Data Warrior and ECOSAR tool. Further refinement by autodock program revealed two compounds ZINC49821385 and ZINC97218938 with the best binding energy of -7.07 and -6.79 respectively. These results indicated that 5-(4- isopropylphenyl)furan-2-carboxamide (ZINC ID 49821385) could be one of the potential ligand to treat bleaching disease in algae.

Molecular identification and genetic diversity of Bartonella spp. in 24 bat species from Thailand.

The study of bacterial zoonoses has been under-pursued despite the fact that bacteria cause the majority of zoonotic diseases, of which 70% have a wildlife origin. More Bartonella species are being identified as the cause of human diseases, and several of them have been linked to domestic and wild animals. Bats are outstanding reservoirs for Bartonella species because of their wide distribution, mobility, roosting behaviour, and long life span. Here, we carried out a PCR-based survey on bats that were collected from 19 sampling sites in eight provinces of Thailand from February 2018 to April 2021. Bartonella infection was investigated in a total of 459 bats that belong to 24 different bat species (21 species of which had never been previously studied in Thailand). PCR diagnostics revealed that 115 out of 459 (25.5%) blood samples tested positive for Bartonella. The nucleotide identities of the Bartonella 16S rRNA sequences in this study were between 95.78-99.66% identical to those of known zoonotic species (Bartonella ancashensis, Bartonella henselae, Bartonella bacilliformis and Bartonella australis) as well as to an unidentified Bartonella spp. In addition, the citrate synthase (gltA) and RNA polymerase-beta subunit (rpoB) genes of Bartonella were sequenced and analyzed in positive samples. The gltA and rpoB gene sequences from Hipposideros gentilis and Rhinolophus coelophyllus bat samples showed low nucleotide identity (<95%) compared to those of the currently deposited sequences in the GenBank database, indicating the possibility of new Bartonella species. The phylogenetic inference and genetic diversity were generated and indicated a close relationship with other Bartonella species previously discovered in Asian bats. Overall, the current study demonstrates the primary evidence pointing to a potential novel Bartonella species in bats. This discovery also contributes to our current understanding of the geographical distribution, genetic diversity, and host ranges of bat-related Bartonella.

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.

Identification and Characterization of Pleiotropic High-Persistence Mutations in the Beta Subunit of the Bacterial RNA Polymerase.

Mutations conferring resistance to bactericidal antibiotics reduce the average susceptibility of mutant populations. It is unknown, however, how those mutations affect the survival of individual bacteria. Since surviving bacteria can be a reservoir for recurring infections, it is important to know how survival rates may be affected by resistance mutations and by the choice of antibiotics. Here, we present evidence that (i) Escherichia coli mutants with 100 to 1,000 times increased frequency of survival in ciprofloxacin, an archetypal fluoroquinolone antibiotic, can be readily obtained in a stepwise selection; (ii) the high survival frequency is conferred by mutations in the switch region of the beta subunit of the RNA polymerase; (iii) the switch-region mutations are (p)ppGpp mimics, partially analogous to rpoB stringent mutations; (iv) the stringent and switch region rpoB mutations frequently occur in clinical isolates of E. coli, Acinetobacter baumannii, Mycobacterium tuberculosis, and Staphylococcus aureus, and at least one of them, RpoB S488L, which is a common rifampicin resistance mutations, dramatically increases the survival of a clinical methicillin-resistant S. aureus (MRSA) strain in ampicillin; and (v) the RpoB-associated high-survival phenotype can be reversed by subinhibitory concentrations of chloramphenicol.

Biomolecular Investigation of Bartonella spp. in Wild Rodents of Two Swiss Regions.

Rodents represent a natural reservoir of several Bartonella species, including zoonotic ones. In this study, small wild rodents, collected from two sites in rural areas of Switzerland, were screened for Bartonella spp. using molecular detection methods. In brief, 346 rodents were trapped in two rural sites in the Gantrisch Nature Park of Switzerland (Plasselb, canton of Fribourg, and Riggisberg, canton of Bern). Pools of DNA originating from three animals were tested through a qPCR screening and an end-point PCR, amplifying the 16S-23S rRNA gene intergenic transcribed spacer region and citrate synthase (gltA) loci, respectively. Subsequently, DNA was extracted from spleen samples belonging to single animals of gltA positive pools, and gltA and RNA polymerase subunit beta (rpoB) were detected by end-point PCR. Based on PCR results and sequencing, the prevalence of infection with Bartonella spp. in captured rodents, was 21.10% (73/346): 31.78% in Apodemus sp. (41/129), 10.47% in Arvicola scherman (9/86), 17.05% in Myodes glareolus (22/129), and 50% in Microtus agrestis (1/2). A significant association was observed between Bartonella spp. infection and rodent species (p < 0.01) and between trapping regions and positivity to Bartonella spp. infection (p < 0.001). Similarly, prevalence of Bartonella DNA was higher (p < 0.001) in rodents trapped in woodland areas (66/257, 25.68%) compared to those captured in open fields (9/89, 10.11%). Sequencing and phylogenetic analysis demonstrated that the extracted Bartonella DNA belonged mainly to B. taylorii and also to Candidatus “Bartonella rudakovii”, B. grahamii, B. doshiae, and B. birtlesii. In conclusion, the present study could rise public health issues regarding Bartonella infection in rodents in Switzerland.

Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., low salt-tolerant haloarchaea isolated from seawater and sediment.

Three novel halophilic archaea were isolated from seawater and sediment near Yeoungheungdo Island, Republic of Korea. The genome size and G + C content of the isolates MBLA0076T, MBLA0077T, and MBLA0078T were 3.56, 3.48, and 3.48Mb and 61.7, 60.8, and 61.1mol%, respectively. The three strains shared 98.5-99.5 % sequence similarity of the 16S rRNA gene, whereas their sequence similarity to the 16S rRNA gene of type strains was below 98.5 %. Phylogenetic analysis based on sequences of the 16S rRNA and RNA polymerase subunit beta genes indicated that the isolates belonged to the genus Haloferax. The orthologous average nucleotide identity, average amino-acid identity, and in silico DNA-DNA hybridization values were below species delineation thresholds. Pan-genomic analysis indicated that the three novel strains and 11 reference strains had 8981 pan-orthologous groups in total. Fourteen Haloferax strains shared 1766 core pan-genome orthologous groups, which were mainly related to amino acid transport and metabolism. Cells of the three isolates were gram-negative, motile, red-pink pigmented, and pleomorphic. The strains grew optimally at 30°C (MBLA0076T) and 40°C (MBLA0077T, MBLA0078T) in the presence of 1.28M (MBLA0077T) and 1.7M (MBLA0076T, MBLA0078T) NaCl and 0.1M (MBLA0077T), 0.2M (MBLA0076T), and 0.3M (MBLA0078T) MgCl2·6H2O at pH 7.0-8.0. Cells of all isolates lysed in distilled water; the minimum NaCl concentration necessary to prevent lysis was 0.43M. The major polar lipids of the three strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and sulphated diglycosyl archaeol-1. Based on their phenotypic and genotypic properties, MBLA0076T, MBLA0077T, and MBLA0078T were described as novel species of Haloferax, for which we propose the names Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., respectively. The respective type strains of these species are MBLA0076T (= KCTC 4288T = JCM 34,169T), MBLA0077T (= KCTC 4289T = JCM 34,170T), and MBLA0078T (= KCTC 4290T = JCM 34,171T).

Developing species-specific quantitative real-time polymerase chain reaction primers for detecting &lt;i&gt;Lautropia mirabilis&lt;/i&gt;

This study aimed to develop Lautropia mirabilis -specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis . The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/ RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

The Spectrum of Spontaneous Rifampin Resistance Mutations in the Bacillus subtilis rpoB Gene Depends on the Growth Environment.

Results from previous investigations into spontaneous rifampin resistance (Rifr) mutations in the Bacillus subtilis rpoB gene suggested that the spectrum of mutations depends on the growth environment. However, these studies were limited by low sample numbers, allowing for the potential distortion of the data by the presence of "jackpot" mutations that may have arisen early in the growth of a population. Here, we addressed this issue by performing fluctuation analyses to assess both the rate and spectrum of Rifr mutations in two distinct media: LB, a complete laboratory medium, and SMMAsn, a minimal medium utilizing l-asparagine as the sole carbon source. We cultivated 60 separate populations under each growth condition and determined the mutation rate to Rifr to be slightly but significantly higher in LB cultures. We then sequenced the relevant regions of rpoB to map the spectrum of Rifr mutations under each growth condition. We found a distinct spectrum of mutations in each medium; LB cultures were dominated by the H482Y mutation (27/53 or 51%), whereas SMMAsn cultures were dominated by the S487L mutation (24/51 or 47%). Furthermore, we found through competition experiments that the relative fitness of the S487L mutant was significantly higher in SMMAsn than in LB medium. We therefore conclude that both the spectrum of Rifr mutations in the B. subtilis rpoB gene and the fitness of resulting mutants are influenced by the growth environment. IMPORTANCE The rpoB gene encodes the beta subunit of RNA polymerase, and mutations in rpoB are key determinants of resistance to the clinically important antibiotic rifampin. We show here that the spectrum of mutations in Bacillus subtilis rpoB depends on the medium in which the cells are cultivated. The results show that the growth environment not only plays a role in natural selection and fitness but also influences the probability of mutation at particular bases within the target gene.

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.