Beta Subunit Of RNA Polymerase Research Articles

An Additional L451G452N453 in the RpoB Protein Suppressed the Synthetic Lethality in Escherichia coli at 37 Degrees Caused by Depletion of DnaK/J and Trigger Factor.

Escherichia coli depletion of chaperone trigger factor and DnaK/J were not viable at 37°C, but viable below 30°C. Among the engineered E. coli depleted of trigger factor and DnaK/J, one strain Z625, exhibited survival at 37°C, while another strain Z629 only survived below 30°C. Comparative analysis of fatty acid profiles of Z625 and Z629 revealed absence of numerous saturated fatty acids in Z625 as compared to the wild-type E. coli BW25113. In addition, increased unsaturated fatty acids were present in Z625, whereas the fatty acids profile of Z629 closely resembled that of BW25113. Whole genome sequencing revealed a 9-bp insertion in rpoB of Z625. Combined structural analysis of simulated RpoB protein bearing the amino acid sequence L451G452N453 insertion and susceptibility analysis to rifampicin suggested that the insertion did not disturb the individual RpoB structure as beta subunit of RNA polymerase. Comparative transcriptomic analysis of Z625 and Z629 suggested that this insertion impacted transcription of the overall RNA polymerase in Z625, leading to potential repression of some genes whose overexpression was toxic to E. coli. Additionally, Z625 exhibited distinctive metabolic adaptations, likely contributing to its survival at 37°C. In summary, our study elucidated one LGN insertion in rpoB that impacts transcriptional regulation in E. coli, thereby explaining the survival of E. coli depletion of trigger factor and DnaK/J at 37°C, and these founding suggested that some simple mutations in critical genes like rpoB might play an important role in driving adaptive evolution.

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.

Comprehensive genomics reveals novel sequence types of multidrug resistant Klebsiella oxytoca with uncharacterized capsular polysaccharide K- and lipopolysaccharide O-antigen loci from the National Hospital of Uganda

The Klebsiella oxytoca complex comprises diverse opportunistic bacterial pathogens associated with hospital and community-acquired infections with growing alarming antimicrobial resistance. We aimed to uncover the genomic features underlying the virulence and antimicrobial resistance of isolates from Mulago National Hospital in Uganda. We coupled whole genome sequencing with Pathogenwatch multilocus sequence typing (MLST) and downstream bioinformatic analysis to delineate sequence types (STs) capsular polysaccharide K- and O-antigen loci, along with antimicrobial resistance (AMR) profiles of eight clinical isolates from the National Referral Hospital of Uganda. Our findings revealed that only two isolates (RSM6774 and RSM7756) possess a known capsular polysaccharide K-locus (KL74). The rest carry various unknown K-loci (KL115, KL128, KLI52, KL161 and KLI63). We also found that two isolates possess unknown loci for the lipopolysaccharide O-antigen (O1/O2v1 type OL104 and unknown O1). The rest possess known O1 and O3 serotypes. From MLST, we found four novel sequence types (STs), carrying novel alleles for the housekeeping genes glyceraldehyde-6-phosphate dehydrogenase A (gapA), glucose-6-phosphate isomerase (pgi), and RNA polymerase subunit beta (rpoB). Our AMR analysis revealed that all the isolates are resistant to ampicillin and ceftriaxone, with varied resistance to other antibiotics, but all carry genes for extended-spectrum beta-lactamases (ESBLs). Notably, one strain (RSM7756) possesses outstanding chromosomal and plasmid-encoded AMR to beta-lactams, cephalosporins, fluoroquinolones and methoprims. Conclusively, clinical samples from Mulago National Referral Hospital harbor novel STs and multidrug resistant K. oxytoca strains, with significant public health importance, which could have been underrated.

Bartonella species in dromedaries and ruminants from Lower Shabelle and Benadir regions, Somalia.

Bartonellosis, caused by bacteria of the genus Bartonella, is a zoonotic disease with several mammalian reservoir hosts. In Somalia, a country heavily reliant on livestock, zoonotic diseases pose significant public health and economic challenges. To the best of our knowledge, no study has been performed aiming to verify the occurrence of Bartonella spp. in Somalia. This study investigated the occurrence and molecular characterization of Bartonella in dromedary (Camelus dromedarius, Linnaeus, 1758), cattle, sheep, and goats from Somalia. 530 blood samples were collected from various animals (155 dromedary, 199 goat, 131 cattle, and 45 sheep) in Benadir and Lower Shabelle regions. DNA was extracted for molecular analysis, and a qPCR assay targeting the NADH dehydrogenase gamma subunit (nuoG) gene was used for Bartonella screening. Positive samples were also subjected to PCR assays targeting seven molecular markers including: nuoG, citrate synthase gene (gltA), RNA polymerase beta-subunit gene (rpoB), riboflavin synthase gene (ribC), 60 kDa heat-shock protein gene (groEL), cell division protein gene (ftsZ), and pap31 and qPCR targeting the 16-23S rRNA internal transcribed spacer (ITS) followed by Sanger sequencing, BLASTn and phylogenetic analysis. Out of 530 tested animals, 5.1% were positive for Bartonella spp. by the nuoG qPCR assay. Goats showed the highest Bartonella occurrence (17/199, 8.5%), followed by sheep (6/44, 6.8%), cattle (4/131, 3.1%), and dromedary (1/155, 1.9%). Goats, sheep, and cattle had higher odds of infection compared to dromedary. Among nuoG qPCR-positive samples, 11.1%, 14.8%, 11.1%, and 25.9% were positive in PCR assays based on nuoG, gltA, and pap31 genes, and in the qPCR based on the ITS region, respectively. On the other hand, nuoG qPCR-positive samples were negative in the PCR assays targeting the ribC, rpoB, ftsZ, and groEL genes. While Bartonella bovis sequences were detected in cattle (nuoG and ITS) and goats (gltA), Bartonella henselae ITS sequences were detected in dromedary, goat, and sheep. Phylogenetic analysis placed gltA Bartonella sequence from a goat in the same clade of B. bovis. The present study showed, for the first time, molecular evidence of Bartonella spp. in dromedary and ruminants from Somalia and B. henselae in sheep and goats globally. These findings contribute valuable insights into Bartonella spp. occurrence in Somali livestock, highlighting the need for comprehensive surveillance and control measures under the One Health approach.

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.

Complete genome sequences and multidrug resistance genotypes of nontuberculous mycobacteria isolates from the Central Tuberculosis Reference Laboratory Muhimbili Tanzania

Background: Nontuberculous mycobacteria (NTM) usually comprise a group of environmental bacteria, with emerging but elusive coinfection with tuberculous mycobacteria, causing pulmonary tuberculosis. Whole genome sequencing may give insight into potential antimicrobial resistance genotypes, giving clinicians and policymakers proper directions in clinical applications and management regimens. Methods: WGS was performed on twenty-four gDNA isolates from archival samples at the Central Tuberculosis Reference Laboratory using the MinION Oxford Nanopore Sequencing approach. Out of twenty-four, two were confirmed to belong to the NTM group. Further analysis was done to resolve the complete genomes of two nontuberculous mycobacteria strains isolated from tuberculosis patients. We then combined phylogenomics, reference-based scaffolding and average nucleotide identity (ANI) analysis to delineate each strain's taxonomic position and corresponding features. Results: Our findings reveal that the two strains fit into the genus Mycolicibacterium, and the closest relative is Mycolicibacterium novocastrense. Coupling BacAnt and CARD-based antibiotic resistance analyses revealed multidrug-resistant genotypes of diverse spectra and mechanisms. While the BC02 strain is genetically resistant to beta-lactams, macrolides and rifamycins, the BC05 strain portrays an extended drug resistance genotype encompassing beta-lactams, macrolides, polyamines, and aminoglycosides. Both strains possess a single nucleotide polymorphism (SNP) of the RNA polymerase beta-subunit (rpoB), representing resistance to the first-line rifampicin. Additionally, the BC05 strain genetically portrays resistance to ethambutol, isoniazid and fosfomycin through mechanisms involving target alteration through SNPs, drug inactivation and efflux. Conclusion: Our findings strongly suggest the potential implication of multidrug-resistant NTM clinical isolates in the pathogenesis of pulmonary tuberculosis.

First report of Bartonella sp. isolated from Hippobosca equina L.(Hippoboscidae: Hippobosca) in Lublin Province, south-eastern Poland.

Hippobosca equina (Diptera: Hippoboscidae), is a widespread blood-feeding ectoparasite associated with the forest ecosystem. The insect is characterized by a wide host range and low host specificity, which increases the risk of feeding on animals that constitute a reservoir of transmissible pathogens, including Bartonella spp. Hippobosca equina adults were collected from humans and companion animals within a continental mesotrophic oak-pine mixed forest in eastern Poland. DNA was isolated by the ammonia method, and isolates obtained from single individuals were tested by PCR method for the presence of 5 vector-borne pathogens. In case of the positive results, the amplicons were sequenced and examined by a BLAST search. The PCR analysis of DNA isolates obtained from 100 H.equina specimens revealed the presence of the RNA polymerase beta-subunit gene (rpoB) of the genus Bartonella, in 1% of the studied insects, i.e. one H.equina female. The rpoB gene haplotype of Bartonella sp. reported in this study, was identical to a Bartonella sp. sequence obtained from deer keds in Lithuania, and very closely related to strains with zoonotic potential. None of the H.equina specimens studied was positive for the presence of B.burgdorferi s.l., Anaplasma phagocytophilum, Babesia spp., and Coxiella burnetii. The study indicates the need to screen the occurrence of Bartonella spp., both in potential vectors and reservoirs of this pathogen in various habitats.

Core Genome of Deinococcota Phylum from 72 Strains Across 40 Species Consist of Only One Gene, Beta Subunit of DNA-Directed RNA Polymerase

Core Genome of Deinococcota Phylum from 72 Strains Across 40 Species Consist of Only One Gene, Beta Subunit of DNA-Directed RNA Polymerase

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.

First Report of Wilt Disease of Ginger Caused by Achromobacter xylosoxidans in China

Ginger (Zingiber officinale) is an important commercial crop that has been widely cultivated in China for more than 2500 years. One variety, Tongling white ginger, has been grown in the Yi'an District of Tongling city, Anhui province (30°45 N, 117°43 E), China. In August 2022, symptoms of yellowing and wilting were observed on ginger plants, with a disease incidence rate exceeding 20% in the field. The stem base of the diseased plants became soft, brown and water-soaked. Additionally, the rhizomes displayed symptoms of browning and water-soaked rot, resembling those caused by Ralstonia solanacearum and Enterobacter cloacae (Yu et al. 2003; Nishijima et al. 2004; Liu et al. 2021). Consequently, ginger bacterial wilt disease may potentially emerge from a combination of infections by diverse pathogenic bacteria. To identify novel pathogens causing the wilt disease, stem tissues of the diseased plants from different locations were sterilized with 1% sodium hypochlorite (NaOCl) for 10 min, followed by at least three time rinses with sterile water. The sterilized samples were then ground with 0.9% saline solution and left at room temperature for 30 min. A 20 μL aliquot of the suspension was serially diluted and cultured on Luria-Bertani (LB) medium at 28°C. A total of 217 isolates was picked and purified for taxonomic identification by 16S rRNA gene analyses with the primer 27F/1492R (Weisburg et al. 1991). Among these isolates, 23 single colony isolates were identified as A. xylosoxidans through NCBI BLASTn analyses. Furthermore, three single isolates from different locations were randomly selected for further experiments. The growing colonies appeared opaque white and round. Microscopic evaluation revealed that cells were rod-shaped with an average length of 1.95 µm and average width of 0.46 µm. The three isolates shared identical 16S rRNA sequences, displayed 99.72% identity with the sequence from A. xylosoxidans strain SeqID2 (GenBank accession NO. MH266081.1). The glutamate synthase (gltB), GTP-binding membrane protein (lepA), NADH:ubiquinone oxidoreductase subunit L (nuoL), RNA polymerase beta-subunit (rpoB), and the enolase (eno) genes of the three isolates were amplified by PCR using primer pairs gltB-F/gltB-R, lepA-F/lepA-R, nuoL-F/nuoL-R, rpoB-F/rpoB-R and eno-F/eno-R, respectively (Spilker et al. 2012; Vandamme et al. 2016). The gene sequences of 16S rRNA (OQ711945, OQ740153 and OR230037), gltB (OR242732, OQ737692 and OR262112), lepA (OR233727, OQ737693 and OR262113), nuoL (OR233726, OQ737694 and OR262114), ropB (OR233725, OQ737695 and OR262115) and eno (OR242733, OQ737696 and OR262116) for the isolates ZOR02, ZOR05 and ZOR12 were deposited in GenBank database. The gltB, lepA, nuoL, rpoB and eno sequences of the isolates ZOR02, ZOR05 and ZOR12 showed 98.66-99.16%, 98.9-100%, 96.28%-97.34%, 98.47-99.44% and 99.27-99.82% similarity to A. xylosoxidans strain AX27, respectively. Phylogenetic trees were constructed based on the 16S rRNA and gltB-lepA-nuoL-rpoB-eno multilocus using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in MEGA11.0 software (Álvarez et al. 2018). For pathogenicity tests, bacterial suspensions were initially prepared in sterile water at a final concentration of 108 CFU mL-1. Subsequently, 10 μL of bacterial suspensions was injected into the stem base of two-month-old ginger plants, while sterile water was used as a control (Wang et al. 2022). These plants were then incubated at 28°C and 70% relative humidity. There were three replicates for each treatment, and each replicate contained five plants. After six days of inoculation, the ginger plants injected with bacterial suspensions alone exhibited severe wilting symptoms similar to those observed in the field. However, water-soaked symptoms were not observed on rhizome tissues from the pathogen-infected plants. Bacterial pathogens were re-isolated from the diseased plants and identified using the morphological and molecular methods to meet Koch's hypothetical tests. To our knowledge, this is the first report of A. xylosoxidans causing wilt disease of ginger in China. In 2022, the average yield loss due to wilt disease in the Yi'an District of Tongling exceeded 20%, posing a major threat to local ginger cultivation. Effective disease management strategies are needed to develop for the control and prevention of the disease.

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.

Phylogenetically related soil actinomycetes distinguish isolation sites by their metabolic activities.

Soil environments are inhabited by microorganisms adapted to its diversified microhabitats. The metabolic activity of individual strains/populations reflects resources available at a particular spot, quality of which may not comply with broad soil characteristics. To explore the potential of individual strains to adapt to particular micro-niches of carbon sources, a set of 331 Actinomycetia strains were collected at ten sites differing in vegetation, soil pH, organic matter content and quality. The strains were isolated on the same complex medium with neutral pH and their metabolites analyzed by UHPLC and LC-MS/MS in spent cultivation medium (metabolic profiles). For all strains, their metabolic profiles correlated with soil pH and organic matter content of the original sites. In comparison, strains phylogeny based on either 16S rRNA or the beta-subunit of DNA-dependent RNA polymerase (rpoB) genes was partially correlated with soil organic matter content but not soil pH at the sites. Antimicrobial activities of strains against Kocuria rhizophila, Escherichia coli, and Saccharomyces cerevisiae were both site- and phylogeny-dependent. The precise adaptation of metabolic profiles to overall sites characteristics was further supported by the production of locally specific bioactive metabolites and suggested that carbon resources represent a significant selection pressure connected to specific antibiotic activities.

Elucidating the molecular mechanisms underlying mutations in Mycobacterium tuberculosis RNA polymerase that confer resistance to rifampicin and its structural analogues

Tuberculosis (TB) is a major global health problem caused by Mycobacterium tuberculosis (Mtb), and is responsible for significant morbidity and mortality worldwide. Rifampicin and its structural analogues are essential first-line anti-TB drugs that inhibit RNA synthesis by binding to the beta-subunit of Mtb RNA polymerase (RNAP). However, the emergence of rifampicin resistant Mtb strains poses a major challenge for TB control efforts. Mutations in the rpoB gene encoding the beta-subunit of RNAP are the most common cause of rifampicin resistance in Mtb. Understanding the molecular mechanisms underlying these mutations and their effects on RNAP function is crucial for developing new drugs and combination therapies to overcome rifampicin resistance in Mtb. This review discusses the molecular mechanisms underlying rifampicin resistance in Mtb RNAP, including the genetic basis and identification of mutations. It can be hypothesized that rifampicin resistance in Mtb RNAP is a multifactorial phenomenon involving structural, biochemical, and genetic factors. The review highlights strategies for developing new drugs and combination therapies to overcome rifampicin resistance in Mtb and future directions for research on the molecular mechanisms underlying rifampicin resistance in Mtb RNAP.

First report of Arthrobacter woluwensis causing brown blotch disease on white button mushroom (Agaricus bisporus).

Agaricus bisporus is one of the most commonly grown edible fungi in the world. In December 2021, brown blotch disease (2% incidence) was observed on the cap of A. bisporus, growing in a mushroom cultivation base in Guangxi, China. Initially, brown blotches (1-1.3 cm) appeared on the cap of A. bisporus, which expanded gradually as the cap grew. After two days, the infection penetrated inner tissues of fruiting bodies, and blotches were dark brown. For the isolation of causative agent(s), internal tissue samples of the infected stipes (5×5×5 mm) were sterilized in 75% ethanol for 30 s, rinsed three times with sterile deionized water (SDW), then, mashed in the sterile 2 ml Eppendorf tubes, 1000 µl SDW was added and the suspension was diluted into seven concentrations (10-1~10-7). Each suspension (120 µl) was spread on Luria Bertani (LB) medium and incubated for 24 hours at 28 °C. Morphological examination of the isolates was referred to Liu et al (2022). The dominant single colonies were whitish-grayish, smooth, convex. The cells were Gram-positive, non-flagellated, nonmotile, no pods or endospores formed, and no fluorescent pigments production on King's B medium (Solarbio). Amplified 16S rRNA (1351 bp; OP740790) of five colonies using universal primers 27f/1492r (Liu et al., 2022), exhibited 99.26% identity with Arthrobacter (Ar.) woluwensis. The partial sequences of the ATP synthase subunit beta gene (atpD) (677 bp; OQ262957), RNA polymerase subunit beta gene (rpoB) (848 bp; OQ262958), preprotein translocase subunit SecY gene (secY) (859 bp; OQ262959) and elongation factor Tu gene (tuf) (831 bp; OQ262960) genes of colonies were amplified using the method of Liu et al (2018), also exhibited more than 99% similarities to Ar. woluwensis. The biochemical tests for isolates (n=3) were performed via bacterial micro-biochemical reaction tubes (Hangzhou Microbial Reagent Co., LTD), and the results showed the same biochemical characteristics as Ar. woluwensis (Positive for esculin hydrolysis, urea, gelatinase, catalase, sorbitol, gluconate, salicin and arginine. Negative for citrate, nitrate reduction and rhamnose) (Funke et al., 1996). The isolates were identified as Ar. woluwensis based on morphological characteristics, biochemical tests and phylogenetic analysis. Pathogenicity tests were performed with bacterial suspensions (1 × 109 CFU/ml) after growing for 36 h in LB Broth at 28 °C, 160 rpm. 30 µl bacterial suspension was added to the cap and tissue of young A. bisporus. SDW was added as a negative control. All treatments were incubated at 20 °C and 80-85% humidity. The experiment was repeated three times with five caps and five tissues of young A. bisporus each time. Brown blotches were observed on all the parts of the inoculated caps and tissues after 24 h of inoculation. At 48 h, the inoculated caps turned dark brown while the infected tissues changed from brown to black and expanded to the entire tissue block giving a severely rotten appearance and foul odor. This disease symptoms were similar to those observed in the original samples. There were no lesions in the control group. After the pathogenicity test, the pathogen was re-isolated from the infected caps and tissues based on morphological characteristics, 16S rRNA sequences, and biochemical results, fulfilling Koch's postulates. Arthrobacter spp. are very widely distributed in the environment (Kim et al., 2008). To date, two studies have confirmed Arthrobacter spp. as a pathogen of edible fungi (Bessette, 1984; Wang et al., 2019). However, this is the first report of Ar. woluwensis causing brown blotch disease on A. bisporus. Our finding could contribute to developing phytosanitary and control treatments for this disease.

Abundant resistome determinants in rhizosphere soil of the wild plant Abutilon fruticosum

A metagenomic whole genome shotgun sequencing approach was used for rhizospheric soil micribiome of the wild plant Abutilon fruticosum in order to detect antibiotic resistance genes (ARGs) along with their antibiotic resistance mechanisms and to detect potential risk of these ARGs to human health upon transfer to clinical isolates. The study emphasized the potential risk to human health of such human pathogenic or commensal bacteria, being transferred via food chain or horizontally transferred to human clinical isolates. The top highly abundant rhizospheric soil non-redundant ARGs that are prevalent in bacterial human pathogens or colonizers (commensal) included mtrA, soxR, vanRO, golS, rbpA, kdpE, rpoB2, arr-1, efrA and ileS genes. Human pathogenic/colonizer bacteria existing in this soil rhizosphere included members of genera Mycobacterium, Vibrio, Klebsiella, Stenotrophomonas, Pseudomonas, Nocardia, Salmonella, Escherichia, Citrobacter, Serratia, Shigella, Cronobacter and Bifidobacterium. These bacteria belong to phyla Actinobacteria and Proteobacteria. The most highly abundant resistance mechanisms included antibiotic efflux pump, antibiotic target alteration, antibiotic target protection and antibiotic inactivation. antimicrobial resistance (AMR) families of the resistance mechanism of antibiotic efflux pump included resistance-nodulation-cell division (RND) antibiotic efflux pump (for mtrA, soxR and golS genes), major facilitator superfamily (MFS) antibiotic efflux pump (for soxR gene), the two-component regulatory kdpDE system (for kdpE gene) and ATP-binding cassette (ABC) antibiotic efflux pump (for efrA gene). AMR families of the resistance mechanism of antibiotic target alteration included glycopeptide resistance gene cluster (for vanRO gene), rifamycin-resistant beta-subunit of RNA polymerase (for rpoB2 gene) and antibiotic-resistant isoleucyl-tRNA synthetase (for ileS gene). AMR families of the resistance mechanism of antibiotic target protection included bacterial RNA polymerase-binding protein (for RbpA gene), while those of the resistance mechanism of antibiotic inactivation included rifampin ADP-ribosyltransferase (for arr-1 gene). Better agricultural and food transport practices are required especially for edible plant parts or those used in folkloric medicine.

Simultaneous detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacteria coexisting in patients with pulmonary tuberculosis by single-tube multiplex PCR.

In clinical diagnostics, molecular methods are used to detect Mycobacterium tuberculosis bacilli (MTB) and to distinguish them from non-tuberculous mycobacteria (NTM). They are also used to make the right treatment decision for the patient as soon as possible. The aim of this study was to establish a rapid and novel multiplex PCR (mPCR) assay for the detection and differentiation of MTB and NTM in a single tube. 100 sputum samples positive for acid-fast bacilli (AFB) were included in this study. Mycobacterial culture, biochemical tests, and antibiotic susceptibility testing were performed on samples. After alkaline decontamination, total DNA was extracted from the samples. A primer pair targeting the rpoB gene, encoding the beta-subunit of RNA polymerase, was used to detect MTB and NTM, amplifying a 235-bp fragment of MTB and a 136-bp sequence of NTM. A pair of primers targeting a 190-bp fragment of the IS6110 region of MTB was also used to confirm the results. The sensitivity and specificity of the mPCR assay were evaluated using DNA extracted from standard strains. The amplified products were then analyzed by conventional agarose gel electrophoresis. Of 100 AFB smear-positive sputum samples, 92 MTB DNA, 7 NTM DNA, and one mixed-infection sample were identified in a single tube using mPCR assay. There was no correlation between the AFB degree of smear positivity and PCR results. Of seven NTM isolates, 6 (86%) were resistant to rifampin, isoniazid, and ethambutol, the three first-line anti-tuberculosis drugs. A single-tube mPCR assay based on the rpoB gene provides a rapid and reliable means of detecting and differentiating MTB and NTM in sputum specimens.

Phenotypic and genotypic adaptation of Escherichia coli to thermal stress is contingent on genetic background.

Evolution can be contingent on history, but we do not yet have a clear understanding of the processes and dynamics that govern contingency. Here we performed the second phase of a two-phase evolution experiment to investigate features of contingency. The first phase of the experiment was based on Escherichia coli clones that had evolved at the stressful temperature of 42.2°C. The Phase 1 lines generally evolved through two adaptive pathways: mutations of rpoB, which encodes the beta subunit of RNA polymerase, or through rho, a transcriptional terminator. We hypothesized that epistatic interactions within the two pathways constrained their future adaptative potential, thus affecting patterns of historical contingency. Using 10 different E. coli Founders representing both adaptive pathways, we performed a second phase of evolution at 19.0°C to investigate how prior genetic divergence or adaptive pathway (rpoB vs. rho) affects evolutionary outcomes. We found that phenotype, as measured by relative fitness, was contingent on founder genotypes and pathways. This finding extended to genotypes, because E. coli from different Phase 1 histories evolved by adaptive mutations in distinct sets of genes. Our results suggest that evolution depends critically on genetic history, likely due to idiosyncratic epistatic interactions within and between evolutionary modules.

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.