Repression of btuB gene transcription in Escherichia coli by the GadX protein

<p>Abstract</p> <p>Background</p> <p>BtuB (B twelve uptake) is an outer membrane protein of <it>Escherichia coli</it>, it serves as a receptor for cobalamines uptake or bactericidal toxin entry. A decrease in the production of the BtuB protein would cause <it>E. coli </it>to become resistant to colicins. The production of BtuB has been shown to be regulated at the post-transcriptional level. The secondary structure switch of 5' untranslated region of <it>butB </it>and the intracellular concentration of adenosylcobalamin (Ado-Cbl) would affect the translation efficiency and RNA stability of <it>btuB</it>. The transcriptional regulation of <it>btuB </it>expression is still unclear.</p> <p>Results</p> <p>To determine whether the <it>btuB </it>gene is also transcriptionally controlled by trans-acting factors, a genomic library was screened for clones that enable <it>E. coli </it>to grow in the presence of colicin E7, and a plasmid carrying <it>gadX </it>and <it>gadY </it>genes was isolated. The <it>lacZ </it>reporter gene assay revealed that these two genes decreased the <it>btuB </it>promoter activity by approximately 50%, and the production of the BtuB protein was reduced by approximately 90% in the presence of a plasmid carrying both <it>gadX </it>and <it>gadY </it>genes in <it>E. coli </it>as determined by Western blotting. Results of electrophoretic mobility assay and DNase I footprinting indicated that the GadX protein binds to the 5' untranslated region of the <it>btuB </it>gene. Since <it>gadX </it>and <it>gadY </it>genes are more highly expressed under acidic conditions, the transcriptional level of <it>btuB </it>in cells cultured in pH 7.4 or pH 5.5 medium was examined by quantitative real-time PCR to investigate the effect of GadX. The results showed the transcription of <it>gadX </it>with 1.4-fold increase but the level of <it>btuB </it>was reduced to 57%.</p> <p>Conclusions</p> <p>Through biological and biochemical analysis, we have demonstrated the GadX can directly interact with <it>btuB </it>promoter and affect the expression of <it>btuB</it>. In conclusion, this study provides the first evidence that the expression of <it>btuB </it>gene is transcriptionally repressed by the acid responsive genes <it>gadX </it>and <it>gadY</it>.</p

A comprehensive survey of the analytical, numerical and experimental methodologies for dynamics of multibody mechanical systems with clearance or imperfect joints

"Available online 19 December 2017"A comprehensive survey of the literature of the most relevant analytical, numerical, and experimental approaches for the kinematic and dynamic analyses of multibody mechanical systems with clearance joints is presented in this review. Both dry and lubricated clearance joints are addressed here, and an effort is made to include a large number of research works in this particular field, which have been published since the 1960′s. First, the most frequently utilized methods for modeling planar and spatial multibody mechanical systems with clearance joints are analyzed, and compared. Other important phenomena commonly associated with clearance joint models, such as wear, non-smooth behavior, optimization and control, chaos, and uncertainty and links’ flexibility, are then discussed. The main assumptions procedures and conclusions for the different methodologies are also examined and compared. Finally, future developments and new applications of clearance joint modeling and analysis are highlighted.This research was supported in part by the China 111 Project (B16003) and the National Natural Science Foundation of China under Grants 11290151, 11472042 and 11221202. The work was also supported by the Portuguese Foundation for Science and Technology with the reference project UID/EEA/04436/2013, by FEDER funds through the COMPETE 2020 – Programa Operacional Competitividade e Internacionalização (POCI) with the reference project POCI-01-0145-FEDER-006941.info:eu-repo/semantics/publishedVersio

Activation of an NLRP3 Inflammasome Restricts Mycobacterium kansasii Infection

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, M. kansasii induced caspase-1-dependent IL-1β secretion in macrophages. The underlying mechanism was found to be through activation of an inflammasome containing the NLR (Nod-like receptor) family member NLRP3 and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Further, potassium efflux, lysosomal acidification, ROS production and cathepsin B release played a role in M. kansasii-induced inflammasome activation. Finally, the secreted IL-1β derived from caspase-1 activation was shown to restrict intracellular M. kansasii. These findings demonstrate a biological role for the NLRP3 inflammasome in host defense against M. kansasii

Mab_3168c, a putative acetyltransferase, enhances adherence, intracellular survival and antimicrobial resistance of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium. It can cause diseases in both immunosuppressed and immunocompetent patients and is highly resistant to multiple antimicrobial agents. M. abscessus displays two different colony morphology types: smooth and rough morphotypes. Cells with a rough morphotype are more virulent. The purpose of this study was to identify genes responsible for M. abscessus morphotype switching. With transposon mutagenesis, a mutant with a Tn5 inserted into the promoter region of the mab_3168c gene was found to switch its colonies from a rough to a smooth morphotype. This mutant had a higher sliding motility but a lower ability to form biofilms, aggregate in culture, and survive inside macrophages. Results of bioinformatic analyses suggest that the putative Mab_3168c protein is a member of the GCN5-related N-acetyltransferase superfamily. This prediction was supported by the demonstration that the mab_3168c gene conferred M. abscessus and M. smegmatis cells resistance to amikacin. The multiple roles of mab_3168c suggest that it could be a potential target for development of therapeutic regimens to treat diseases caused by M. abscessus

Combined <it>rpo</it>B duplex PCR and <it>hsp65</it> PCR restriction fragment length polymorphism with capillary electrophoresis as an effective algorithm for identification of Mycobacterial species from clinical isolates

Abstract Background Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA), but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA) and hsp65 PRA with capillary electrophoresis. Results Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC) and 176 non-tuberculous mycobacteria (NTM) were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100% (200 isolates) for the MTC and 91.4% (161 isolates) for the NTM. Among the discordant results (8.6%) for the NTM, one isolate of Mycobacterial species and an isolate of M. flavescens were found as new sub-types in hsp65 PRA. Conclusions This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16 S rDNA sequencing.</p

Biofilm-forming capability of <i>M. abscessus</i> cells.

<p>(A) Cultures of wild type, <i>mab_3168c</i>::Tn, and complemented strains were grown on 96-well PVC plates for 6 days. The plates were washed and then stained with crystal violet. (B) Optical density readings of the three strains shown are means ± standard deviations of three independent experiments. Data were analyzed by one-way ANOVA and Fisher’s PLSD test. The asterisk sign (*) represents p<0.05 of <i>mab_3168c</i>::Tn versus wild type or <i>mab_3168c</i>::Tn versus complemented strain. (C) Growth rates of <i>M. abscessus</i> variants. Cells were grown in 7H9 medium supplemented with OADC (10%) and Tween 80 (0.05%), and the OD600 value of each culture was measured at the indicated time points.</p

Amikacin E-test.

<p>Amikacin E-test.</p

Identification of <i>mab_3168c</i>::Tn mutant.

<p>(A) Colony morphology of wild type, <i>mab_3168c</i>::Tn, and complemented strains of <i>M. abscessus</i>. Five days old colonies of <i>M. abscessus</i> on 7H11 agar were viewed from the top of the colonies, demonstrating rough and smooth morphologies. (B) Schematic representation of the <i>mab_3168c</i> locus and the inserted EZ-Tn<i>5</i>™ R> transposon (EPICENTRE®). Numbers shown on top of the figure are nucleotide positions of the <i>M. abscessus</i> genome. Primer KAN-2-FP-1 was used to sequence the insertion junctions of Tn<i>5</i>. (C) mRNA levels of <i>mab_3168c</i> and <i>ispG</i> of wild type, <i>mab_3168c</i>::Tn mutant, and complemented strains. The 16S rRNA levels were used as the internal control. The 16S rRNA levels determined without reverse transcription served as the negative control for contamination from chromosomal DNA. This experiment was repeated two times. (D) The intensities of the <i>mab_3168c</i> mRNA and the 16S rRNA bands were determined by densitometry. The relative intensity value of each sample was obtained by dividing the intensity value of its <i>mab_3168c</i> mRNA band by that of its 16S rRNA band. The arbitrary unit of the <i>mab_3168c</i> mRNA band of each strain was calculated as the relative density value of its <i>mab_3168c</i> mRNA band divided by that of the wild type.</p