[Effects of Three Commonly Used Herbicides on Bacterial Antibiotic Resistance].

The spread of antibiotic resistance has become a serious threat to global public health. Recently, several studies have found that non-antibiotic chemicals can promote the generation and spread of bacterial antibiotic resistance. However, the effects of herbicides on the antibiotic resistance of bacteria remain unclear. In this study, Escherichia coli DH5α was used as the model strain to explore the effects of three commonly used herbicides (glyphosate, glufosinate, and dicamba) on the antibiotic resistance under soil environmental concentrations. The results showed that herbicide exposure affected the sensitivity of E. coli DH5α to antibiotics and significantly improved the resistance of E. coli DH5α to gentamicin (glyphosate > dicamba > glufosinate). After 30 d of herbicide exposure, the E. coli mutant strains enhanced the resistance to tetracycline, chloramphenicol, and aminoglycoside antibiotics, and the minimum inhibitory concentration of streptomycin was increased by 19.8 times. The whole-genome sequencing results illustrated that herbicides induced several previously well-characterized mutations associated with membrane proteins (ompF and papC), fimbriae proteins (yraH), and ribosomes (rpsL) related to antibiotic resistance. Together, the results showed that herbicides can enhance the antibiotic resistance of bacteria via inducing genetic mutations, thereby promoting the potential risk of the spread of antibiotic resistance genes in the environment.

LncRNA BRCAT54 inhibits the tumorigenesis of non-small cell lung cancer by binding to RPS9 to transcriptionally regulate JAK-STAT and calcium pathway genes.

OBJECTIVES: Increasing evidence suggest that long non-coding RNAs (lncRNAs) play critical roles in cancers. However, the expression pattern and underlying mechanisms of lncRNAs in non-small cell lung cancer (NSCLC) remain incompletely understood. This study aimed to elucidate the functions and molecular mechanisms of a certain lncRNA in NSCLC. METHODS: LncRNA microarray was performed to identify differential expressed lncRNAs between pre- and postoperation plasma in NSCLC patients. The expression level of candidate lncRNA in NSCLC tissues, plasma and cells was determined by quantitative real-time PCR (qRT-PCR) and in situ hybridization. The functional roles of lncRNA were assessed in vitro and in vivo. Furthermore, RNA pull-down, RNA immunoprecipitation, microarray, qRT-PCR and rescue assays were conducted to explore the mechanism action of lncRNA in NSCLC cells. RESULTS: We identified a novel lncRNA (BRCAT54), which was significantly upregulated in preoperative plasma, NSCLC tissues and NSCLC cells, and its higher expression was associated with better prognosis in patients with NSCLC. Overexpression of BRCAT54 inhibited proliferation, migration and activated apoptosis in NSCLC cells. Conversely, knockdown of BRCAT54 reversed the suppressive effects of BRCAT54. Moreover, overexpression of BRCAT54 repressed NSCLC cell growth in vivo. Mechanistically, BRCAT54 directly bound to RPS9. Knockdown of RPS9 substantially reversed the promoting effects of si-BRCAT54 on cell proliferation and enhanced the inhibitive effect of si-BRCAT54 on BRCAT54 expression. In addition, silencing of RPS9 activated JAK-STAT pathway and suppressed calcium signaling pathway gene expressions. CONCLUSION: This study identified BRCAT54 as a tumor suppressor in NSCLC. Targeting the BRCAT54 and RPS9 feedback loop might be a novel therapeutic strategy for NSCLC.

Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus.

Previously, using FREP-MS, we identified a protein complex including eight proteins that specifically bind to the functional SNP (fSNP) rs6032664 at a CD40 locus associated with autoimmune diseases. Among these eight proteins, four are ribosomal proteins RPL26, RPL4, RPL8, and RPS9 that normally make up the ribosomal subunits involved in the cellular process of protein translation. So far, no publication has shown these ribosomal proteins function as transcriptional regulators. In this work, we demonstrate that four ribosomal proteins: RPL26, RPL4, RPL8, and RPS9 are bona fide CD40 transcriptional regulators via binding to rs6032664. In addition, we show that suppression of CD40 expression by RPL26 RNAi knockdown inactivates NF-κB p65 by dephosphorylation via NF-κB signaling pathway in fibroblast-like synoviocytes (FLS), which further reduces the transcription of disease-associated risk genes such as STAT4, CD86, TRAF1 and ICAM1 as the direct targets of NF-κB p65. Based on these findings, a disease-associated risk gene transcriptional regulation network (TRN) is generated, in which decreased expression of, at least, RPL26 results in the downregulation of risk genes: STAT4, CD86, TRAF1 and ICAM1, as well as the two proinflammatory cytokines: IL1ß and IL6 via CD40-induced NF-κB signaling. We believe that further characterization of this disease-associated TRN in the CD40-induced NF-κB signaling by identifying both the upstream and downstream regulators will potentially enable us to identify the best targets for drug development.

In Vitro and In Vivo Efficacy of DNA Damage Repair Inhibitor Veliparib in Combination with Artesunate against Echinococcus granulosus.

Cystic echinococcosis (CE), caused by the cestode Echinococcus granulosus, is a worldwide chronic zoonosis. Albendazole (ABZ) and mebendazole are effective against CE, but a high dosage in a long-term period is usually required. In this study, we evaluate the effects of DNA damage repair inhibitor (i.e., Veliparib) in combination with artesunate (AS) on hydatid cysts. For the in vitro assay, protoscoleces of E. granulosus (E.g PSCs) were incubated with low AS (AS-L, 65 µM), moderate AS (AS-M, 130 µM), and high AS (AS-H, 325 µM), AS-L/M/H+Veliparib (10 µM), and ABZ (25 µM), respectively. The AS-H+Veliparib group showed the maximal protoscolicidal effects. Ultrastructural change revealed that germinal layer (GL) cells were reduced, and lipid droplets appeared. AS could induce DNA injuries in PSCs. The 8-OHdG was expressed in the PSCs and GL of the cysts in mice, especially in the presence of Veliparib. The most severe DNA damages were observed in the AS-H+Veliparib group. Meanwhile, the expression of ribosomal protein S9 (RPS9) gene in the AS-H+Veliparib group was significantly lower than that in the AS-H group. The in vivo chemotherapeutic effects of AS-L (50 mg/kg), AS-H (200 mg/kg), and AS-H+Veliparib (25 mg/kg) were assessed in experimentally infected mice. Upon 6 weeks of oral administration, ultrasonography was used to monitor the volume change of vesicles. Maximum potentiation was seen on day 15 with values (versus AS) of 34 (P < 0.05) for AS-H + Veliparib. It led to the reduction of cyst weight (55.40%) compared with the model group (P < 0.01), which was better than AS alone (52.84%) and ABZ-treated mice (55.35%). Analysis of cysts collected from AS-H+Veliparib-treated mice by transmission electron microscopy revealed a drug-induced structural destruction. The structural integrity of the germinal layer was lost, and the majority of the microtriches disappeared. In conclusion, our study demonstrates that AS or AS in combination with Veliparib is effective for treating CE, especially the combination group. On this basis, AS represented promising drug candidates in anti-CE chemotherapy.

Radial Expansion Facilitates the Maintenance of Double Antibiotic Resistances.

Most microbes live in spatially confined subpopulations. Under spatial structure conditions, the efficacy of natural selection is often reduced (relative to homogeneous conditions) due to the increased importance of genetic drift and local competition. Additionally, under spatial structure conditions, the fittest genotype may not always be the one with better access to the heterogeneous distribution of nutrients. The effect of radial expansion may be particularly relevant for the elimination of antibiotic resistance mutations, as their dynamics within bacterial populations are strongly dependent on their growth rate. Here, we use Escherichia coli to systematically compare the allele frequency of streptomycin, rifampin, and fluoroquinolone single and double resistance mutants after 24 h of coexistence with a susceptible strain under radial expansion (local competition) and homogeneous (global competition) conditions. We show that there is a significant effect of structure on the maintenance of double resistances which is not observed for single resistances. Radial expansion also facilitates the persistence of double resistances when competing against their single counterparts. Importantly, we found that spatial structure reduces the rate of compensation of the double mutant RpsLK43T RpoBH526Y and that a strongly compensatory mutation in homogeneous conditions becomes deleterious under spatial structure conditions. Overall, our results unravel the importance of spatial structure for facilitating the maintenance and accumulation of multiple resistances over time and for determining the identity of compensatory mutations.

Selection for Antimicrobial Resistance in Foodborne Pathogens through Exposure to UV Light and Nonthermal Atmospheric Plasma Decontamination Techniques.

This study was aimed at assessing whether the repeated exposure of 12 strains of Salmonella spp., Escherichia coli, and Listeria monocytogenes to alternative nonthermal decontamination techniques with UV light (UV-C) and nonthermal atmospheric plasma (NTAP) may cause the emergence of variants showing increased resistance to clinically relevant antibiotics (ampicillin, cefotaxime, ciprofloxacin, gentamicin, streptomycin, tetracycline, erythromycin, vancomycin, and colistin). UV-C and NTAP treatments were applied on the surface of inoculated brain heart infusion (BHI) agar plates. Survivors were recovered and after 24 h of growth in BHI broth were again subjected to the decontamination treatment; this was repeated for 10 consecutive cycles. A total of 174 strain/decontamination technique/antibiotic combinations were tested, and 12 variant strains with increased resistance to one of the antibiotics studied were identified, with the increases in the MICs in Mueller-Hinton broth ranging from 2- to 256-fold. The variant strains of Salmonella spp. isolated were further characterized through phenotypic screenings and whole-genome sequencing (WGS) analyses. Most changes in susceptibility were observed for antibiotics that act at the level of protein synthesis (aminoglycosides, tetracyclines, and glycylcyclines) or DNA replication (fluoroquinolones), as well as for polymyxins. No changes in resistance to ß-lactams were detected. WGS analyses showed the occurrence of sequence alterations in some antibiotic cellular targets (e.g., gyrA for ciprofloxacin-resistant variants, rpsL for a streptomycin-resistant variant), accompanied by variations in stress response regulators and membrane transporters likely involved in the nonselective efflux of antibiotics, which altogether resulted in a low- to medium-level increase in microbial resistance to several antibiotics.IMPORTANCE The emergence and spread of antibiotic resistance along the food chain can be influenced by the different antimicrobial strategies used from farm to fork. This study evidences that two novel, not yet widely used, nonthermal microbial decontamination techniques, UV light and nonthermal atmospheric plasma, can select variants with increased resistance to various clinically relevant antibiotics, such as ciprofloxacin, streptomycin, tetracycline, and erythromycin. Whole-genome analysis of the resistant variants obtained for Salmonella spp. allowed identification of the genetic changes responsible for the observed phenotypes and suggested that some antimicrobial classes are more susceptible to the cross-resistance phenomena observed. This information is relevant, since these novel decontamination techniques are being proposed as possible alternative green techniques for the decontamination of environments and equipment in food and clinical settings.

Loss of rps9 in Zebrafish Leads to p53-Dependent Anemia.

Ribosome is a vital molecular machine for protein translation in the cell. Defects in several ribosomal proteins including RPS19, RPL11 and RPS14 have been observed in two types of anemia: Diamond Blackfan Anemia and 5q- syndrome. In zebrafish, deficiency of these ribosomal proteins shows similar anemic phenotype. It remains to be determined if any other ribosome proteins are similarly involved in regulating erythropoiesis. Here we generated mutations in zebrafish rps9, a rarely studied ribosomal protein gene, and investigated its function. Analysis of this mutant demonstrates that rps9 disruption leads to impairment of erythrocyte maturation, resulting in anemia. In addition, the overall phenotype including the anemic state is p53-dependent in rps9 mutants. Furthermore, this anemic state can be partially relieved by the treatment of L-leucine, and dexamethasone, which have been previously used in rescuing the phenotype of other ribosomal protein mutants. Finally, by comparing the phenotype, we show that there are considerable differences in morphology, cytomorphology, and hemoglobin levels for four ribosomal protein mutants in zebrafish. Based on the observed difference, we suggest that the level of anemic severity correlates with the delayed status of erythrocyte maturation in zebrafish models.

Development of a markerless gene deletion strategy using rpsL as a counterselectable marker and characterization of the function of RA0C_1534 in Riemerella anatipestifer ATCC11845 using this strategy.

Riemerella anatipestifer is a gram-negative bacterium that mainly infects ducks, turkeys and other birds. In a previous study, we established a markerless mutation system based on the pheS mutant as a counterselectable marker. However, the toxic effect of p-Cl-Phe on the R. anatipestifer strain expressing the pheS mutant was weak on blood agar plates. In this study, we successfully obtained streptomycin-resistant derivative of R. anatipestifer ATCC11845 using 100 µg/mL streptomycin as a selection pressure. Then, we demonstrate that rpsL can be used as a counterselectable marker in the R. anatipestifer ATCC11845 rpsL mutant strain, namely, R. anatipestifer ATCCs. A suicide vector carrying wild-type rpsL, namely, pORS, was constructed and used for markerless deletion of the gene RA0C_1534, which encodes a putative sigma-70 family RNA polymerase sigma factor. Using rpsL as a counterselectable marker, markerless mutagenesis of RA0C_1534 was also performed based on natural transformation. R. anatipestifer ATCCsΔRA0C_1534 was more sensitive to H2O2-generated oxidative stress than R. anatipestifer ATCCs. Moreover, transcription of RA0C_1534 was upregulated under 10 mM H2O2 treatment and upon mutation of fur. These results suggest that RA0C_1534 is involved in oxidative stress response in R. anatipestifer. The markerless gene mutation method developed in this study provides new tools for investigation of the physiology and pathogenic mechanisms of this bacterium.

An update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial microorganism, but the availability of tools for its genetic modification has lagged compared to other model microorganisms such as Escherichia coli. Despite great progress in CRISPR-based technologies, the most feasible genome editing method in C. glutamicum is suicide plasmid-mediated, the editing efficiency of which is low due to high false-positive rates of sacB counter selection, and the requirement for tedious two-round selection and verification of rare double-cross-over events. In this study, an rpsL mutant conferring streptomycin resistance was harnessed for counter selection, significantly increasing the positive selection rate. More importantly, with the aid of high selection efficiencies through the use of antibiotics, namely kanamycin and streptomycin, the two-step verification strategy can be simplified to just one-step verification of the final edited strain. As proof of concept, a 2.5-kb DNA fragment comprising aroGfbr pheAfbr expressing cassettes was integrated into the genome of C. glutamicum, with an efficiency of 20% out of the theoretical 50%. The resulting strain produced 110 mg l-1  l-tyrosine in shake-flask fermentation. This updated suicide plasmid-mediated genome editing system will greatly facilitate genetic manipulations including single nucleotide mutation, gene deletion and gene insertion in C. glutamicum and can be easily applied to other microbes.

[Ribosomal Protein S9 Expression in Multiple Myeloma and Its Effect on Cell Biological Function].

Objective To identify the expression of ribosomal protein S9(RPS9)in multiple myeloma(MM)and explore its effect on the biological characteristics of myeloma cells and the corresponding mechanisms. Methods Bone marrow mononuclear cells were harvested in 10 healthy volunteers(CON group)and bone marrow CD138 +cells from 30 MM patients(CD138+group).Quantitative polymerase chain reaction(qPCR)was performed to detect RPS9 expression at mRNA level.In three cases from CON group and 11 cases from CD138+group,Western blot was performed to detect RPS9 at protein level.GSE19784 dataset was employed to detect the relationships of RPS9 expression with the overall survival rate,nuclear factor-κB(NF-κB),small ubiquitin-like modifier(SUMO),and ubiquitin pathway.After the RPS9 knock-down vector was constructed,flow cytometry was performed to detect the infection efficiency and qPCR and Western blot to detect the knock-down efficiency.RPMI8226 was divided into CON group and RPS9-short hairpin RNA(shRNA)group,in which annexin V allophycocyanin/propidium iodide(PI)double staining was performed to detect the change of apoptosis,CCK8 to detect the proliferation change,and PI staining to detect cell cycle change.After sentrin-specific protease 1(SENP1)overexpression vector was constructed,Western blot was performed to detect the phosphorylation of P65 and inhibitory subunit-κBα(IκBα)from NF-κB pathway in CON,RPS9-shRNA,and RPS9-shRNA-SENP1 cells;in addition,annexin V/PI double staining was also performed to detect the apoptosis in these three cells. Results The relative expression of RPS9 in CON group and CD138+group was(1.00±0.12)and(5.45±0.71),respectively(t=4.291,P=0.0036).Western blot showed RPS9 expression was high in most myeloma CD138+cells.The high expression of RPS9 was associated with both extramedullary invasion and overall survival in GSE19784 dataset.After RPMI8226 was infected with CON or RPS9-shRNA lentivirus for 48 hours,flow cytometry confirmed that the infection efficiencies were above 90% in both groups.qPCR and Western blot confirmed that RPS9 expression was inhibited at both mRNA and protein levels.After RPMI8226 CON and RPS9-shRNA infected with virus for 48 hours,the proportion of annexin V-positive cells in CON and RPS9-shRNA cells was(3.47±0.37)% and(18.60±64.00)%(t=9.015,P=0.0008).The proliferation index significantly differed between CON group and RPS9-shRNA group at 72 hours(t=6.846,P=0.0024).When CON and RPS9-shRNA were infected with virus for 48 hours,the proportion of G2 phase cells was(29.28±3.42)% and(10.43±1.43)%,respectively(t=9.329,P=0.0007).The RPS9 expression was positively correlated with SENP1 in GSE19784 dataset and negatively correlated with IκBα coding gene NFKBIA.Western blot further confirmed that RPS9 knockdown inhibited the expression of SENP1,inhibited the phosphorylation of NF-κB subunit P65 and inhibitor IκBα,and promoted the expression of IκBα.Overexpression of SENP1 not only impeded this effect but also reduced RPS9-induced apoptosis. Conclusions RPS9 is highly expressed in MM CD138+cells and is associated with overall survival and extramedullary infiltration.Inhibition of RPS9 can promote apoptosis,cell cycle arrest,and proliferation of myeloma cells.RPS9 can affect the activation of NF-κB pathway and cell apoptosis through SENP1,suggesting that SENP1 may be a key factor in the biological effect of RPS9.

The ribosomal maturation factor P from Mycobacterium smegmatis facilitates the ribosomal biogenesis by binding to the small ribosomal protein S12.

The ribosomal maturation factor P (RimP) is a highly conserved protein in bacteria and has been shown to be important in ribosomal assembly in Escherichia coli Because of its central importance in bacterial metabolism, RimP represents a good potential target for drug design to combat human pathogens such as Mycobacterium tuberculosis However, to date, the only RimP structure available is the NMR structure of the ortholog in another bacterial pathogen, Streptococcus pneumoniae Here, we report a 2.2 Å resolution crystal structure of MSMEG_2624, the RimP ortholog in the close M. tuberculosis relative Mycobacterium smegmatis, and using in vitro binding assays, we show that MSMEG_2624 interacts with the small ribosomal protein S12, also known as RpsL. Further analyses revealed that the conserved residues in the linker region between the N- and C-terminal domains of MSMEG_2624 are essential for binding to RpsL. However, neither of the two domains alone was sufficient to form strong interactions with RpsL. More importantly, the linker region was essential for in vivo ribosomal biogenesis. Our study provides critical mechanistic insights into the role of RimP in ribosome biogenesis. We anticipate that the MSMEG_2624 crystal structure has the potential to be used for drug design to manage M. tuberculosis infections.

Effects of mismatches distant from the target position on gene correction with a 5'-tailed duplex.

The introduction of a 5'-tailed duplex (5'-TD) fragment into cells corrects a base-substitution mutation in a target DNA. We previously reported that the gene correction efficiency was improved when a frameshift type of second mismatch was present ∼330 bases distant from the target position, between the target DNA and the 5'-TD fragment. In this study, the effects of the second mismatches on the gene correction were further examined. Base-base mismatches 332 bases distant from the target position slightly enhanced gene correction, but less efficiently than the previously studied frameshift mismatches. The gene correction efficiency was also increased when the distance between the target position and the second frameshift mismatch was changed to ∼270 bases. These results suggested that the introduction of an appropriate second frameshift mismatch into the 5'-TD fragment improves the gene correction efficiency.

A Novel Tool for Microbial Genome Editing Using the Restriction-Modification System.

Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes on the basis of its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE) technique for scarless genetic manipulation in different microorganisms. For bacteria with Type IV REase, an RMGE technique using the inducible DNA methyltransferase gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE technique based on a restriction endonuclease (RMGE-mcrA) was established in Bacillus subtilis. These techniques were successfully used for gene deletion and replacement with nearly 100% counter-selection efficiencies, which were higher and more stable compared to conventional methods. Furthermore, precise point mutation without limiting sites was achieved in E. coli using RMGE-bceSIIM to introduce a single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414 with 100% counter-selection efficiency. The effectiveness of the RMGE technique in E. coli, B. subtilis, and S. cerevisiae suggests the potential universal usefulness of this technique for microbial genome manipulation.

A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities.

Bacterial recombineering typically relies on genomic incorporation of synthetic oligonucleotides as mediated by Escherichia coli λ phage recombinase ß - an occurrence largely limited to enterobacterial strains. While a handful of similar recombinases have been documented, recombineering efficiencies usually fall short of expectations for practical use. In this work, we aimed to find an efficient Recß homologue demonstrating activity in model soil bacterium Pseudomonas putida EM42. To this end, a genus-wide protein survey was conducted to identify putative recombinase candidates for study. Selected novel proteins were assayed in a standardized test to reveal their ability to introduce the K43T substitution into the rpsL gene of P. putida. An ERF superfamily protein, here termed Rec2, exhibited activity eightfold greater than that of the previous leading recombinase. To bolster these results, we demonstrated Rec2 ability to enter a range of mutations into the pyrF gene of P. putida at similar frequencies. Our results not only confirm the utility of Rec2 as a Recß functional analogue within the P. putida model system, but also set a complete workflow for deploying recombineering in other bacterial strains/species. Implications range from genome editing of P. putida for metabolic engineering to extended applications within other Pseudomonads - and beyond.

Identification of breast cancer hub genes and analysis of prognostic values using integrated bioinformatics analysis.

BACKGROUND: Breast cancer (BC) is the second most common cause of death from cancer in women in the United States. As the molecular mechanism of BC has not yet been completely discovered, identification of hub genes and pathways of this disease is of importance for revealing molecular mechanism of breast cancer initiation and progression. OBJECTIVE: This study aimed to identify potential biomarkers and survival analysis of hub genes for BC treatment. METHODS: The differentially expressed genes (DEGs) between breast cancer and normal cells were screened using microarray data obtained from the Gene Expression Omnibus (GEO) database. Gene ontology (GO) and KEGG pathway enrichment analyses were performed for DEGs using DAVID database, the protein-protein interaction (PPI) network was constructed using the Cytoscape software, and module analysis was performed using MCODE. Then, overall survival (OS) analysis of hub genes was performed by the Kaplan-Meier plotter online tool. Finally, the potential molecular agents were identified with Connectivity Map (cMap) database. RESULTS: A total of 585 DEGs were obtained, which were significantly enriched in the terms related to positive regulation of cell migration, regulation of cell proliferation and focal adhesion. KEGG pathway analysis showed that the significant pathways included Focal adhesion, Pathways in cancer, ECM-receptor interaction, Ribosome, Transcriptional misregulation in cancer and other signaling pathways about cancer. The PPI network was established with 576 nodes and 1943 edges. A significant module was found from the PPI network, the enriched functions and pathways included ECM-receptor interaction and Focal adhesion. CONCLUSIONS: Fifteen genes were selected as hub genes because of high degrees, among which, low expression of four genes was associated with worse OS of patients with BC, including RPS9, RPL11, RPS14 and RPL10A. Additionally, the small molecular agent emetine may be a potential drug for BC.

Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium.

The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA) metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following introduction of solid feed to the weaning neonate as well as affecting the metabolism of other nutrients and absorption of nutrients in in vitro experiments. The objective of the present study was to validate expression stability of eight putative reference genes bovine rumen, considering the intrinsic heterogeneity of bovine rumen with regard to different luminal characteristics due to direct infusion of butyrate to double the intra-ruminal content of the rumen liquor. Our focus was on identifying stable reference genes which are suitable to normalize real-time RT-qPCR experiments from rumen samples collected from clinical assays, irrespective of localization within the organ and the across physiological state. The most stably expressed genes included: ACTB, UXT, DBNDD2, RPS9, DDX54 and HMBS. Their high stability values suggest these reference genes will facilitate better evaluation of variation of across an array of conditions including: localization within the rumen, differences among cattle fed an array of rations, as well as response to development in the weaning animal. Moreover, we anticipate these reference genes may be useful for expression studies in other ruminants.

Streptococcal group B integrative and mobilizable element IMESag-rpsI encodes a functional relaxase involved in its transfer.

Streptococcus agalactiae or Group B Streptococcus (GBS) are opportunistic bacteria that can cause lethal sepsis in children and immuno-compromised patients. Their genome is a reservoir of mobile genetic elements that can be horizontally transferred. Among them, integrative and conjugative elements (ICEs) and the smaller integrative and mobilizable elements (IMEs) primarily reside in the bacterial chromosome, yet have the ability to be transferred between cells by conjugation. ICEs and IMEs are therefore a source of genetic variability that participates in the spread of antibiotic resistance. Although IMEs seem to be the most prevalent class of elements transferable by conjugation, they are poorly known. Here, we have studied a GBS-IME, termed IMESag-rpsI, which is widely distributed in GBS despite not carrying any apparent virulence trait. Analyses of 240 whole genomes showed that IMESag-rpsI is present in approximately 47% of the genomes, has a roughly constant size (approx. 9 kb) and is always integrated at a single location, the 3'-end of the gene encoding the ribosomal protein S9 (rpsI). Based on their genetic variation, several IMESag-rpsI types were defined (A-J) and classified in clonal complexes (CCs). CC1 was the most populated by IMESag-rpsI (more than 95%), mostly of type-A (71%). One CC1 strain (S. agalactiae HRC) was deep-sequenced to understand the rationale underlying type-A IMESag-rpsI enrichment in GBS. Thirteen open reading frames were identified, one of them encoding a protein (MobSag) belonging to the broadly distributed family of relaxases MOBV1 Protein MobSag was purified and, by a newly developed method, shown to cleave DNA at a specific dinucleotide. The S. agalactiae HRC-IMESag-rpsI is able to excise from the chromosome, as shown by the presence of circular intermediates, and it harbours a fully functional mobilization module. Further, the mobSag gene encoded by this mobile element is able to promote plasmid transfer among pneumococcal strains, suggesting that MobSag facilitates the spread of IMESag-rpsI and that this spread would explain the presence of the same IMESag-rpsI type in GBS strains belonging to different CCs.

Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes.

Most budding yeast introns exist in the many duplicated ribosomal protein genes (RPGs) and it has been posited that they remain there to modulate the expression of RPGs and cell growth in response to stress. However, the mechanism by which introns regulate the expression of RPGs and their impact on the synthesis of ribosomal proteins remain unclear. In this study, we show that introns determine the ratio of ribosomal protein isoforms through asymmetric paralog-specific regulation of splicing. Exchanging the introns and 3' untranslated regions of the duplicated RPS9 genes altered the splicing efficiency and changed the ratio of the ribosomal protein isoforms. Mutational analysis of the RPS9 genes indicated that splicing is regulated by variations in the intron structure and the 3' untranslated region. Together these data suggest that preferential splicing of duplicated RPGs provides a means for adjusting the ratio of different ribosomal protein isoforms, while maintaining the overall expression level of each ribosomal protein.

Streptomycin affinity depends on 13 amino acids forming a loop in homology modelled ribosomal S12 protein (rpsL gene) of Lysinibacillus sphaericus DSLS5 associated with marine sponge (Tedania anhelans).

Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms.

Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.