Identification and characterization of smallest pore-forming protein in the cell wall of pathogenic Corynebacterium urealyticum DSM 7109

Abdali N, Younas F, Mafakheri S, et al. Identification and characterization of smallest pore-forming protein in the cell wall of pathogenic Corynebacterium urealyticum DSM 7109. BMC BIOCHEMISTRY. 2018;19(1): 18.Background: Corynebacterium urealyticum, a pathogenic, multidrug resistant member of the mycolata, is known as causative agent of urinary tract infections although it is a bacterium of the skin flora. This pathogenic bacterium shares with the mycolata the property of having an unusual cell envelope composition and architecture, typical for the genus Corynebacterium. The cell wall of members of the mycolata contains channel-forming proteins for the uptake of solutes. Results: In this study, we provide novel information on the identification and characterization of a pore-forming protein in the cell wall of C. urealyticum DSM 7109. Detergent extracts of whole C. urealyticum cultures formed in lipid bilayer membranes slightly cation-selective pores with a single-channel conductance of 1.75 nS in 1 M KCl. Experiments with different salts and non-electrolytes suggested that the cell wall pore of C. urealyticum is wide and water-filled and has a diameter of about 1.8 nm. Molecular modelling and dynamics has been performed to obtain a model of the pore. For the search of the gene coding for the cell wall pore of C. urealyticum we looked in the known genome of C. urealyticum for a similar chromosomal localization of the porin gene to known porH and porA genes of other Corynebacterium strains. Three genes are located between the genes coding for GroEL2 and polyphosphate kinase (PKK2). Two of the genes (cur_1714 and cur_1715) were expressed in different constructs in C. glutamicum Delta porA Delta porH and in porin-deficient BL21 DE3 Omp8 E. coli strains. The results suggested that the gene cur_1714 codes alone for the cell wall channel. The cell wall porin of C. urealyticum termed PorACur was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 4 kDa on tricine-containing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Conclusions: Biophysical characterization of the purified protein (PorACur) suggested indeed that cur_1714 is the gene coding for the pore-forming protein in C. urealyticum because the protein formed in lipid bilayer experiments the same pores as the detergent extract of whole cells. The study is the first report of a cell wall channel in the pathogenic C. urealyticum

Identification of Proximal and Distal 22q11.2 Microduplications among Patients with Cleft Lip and/or Palate: A Novel Inherited Atypical 0.6 Mb Duplication

Misalignments of low-copy repeats (LCRs) located in chromosome 22, particularly band 22q11.2, predispose to rearrangements. A variety of phenotypic features are associated with 22q11.2 microduplication syndrome which makes it challenging for the genetic counselors to recommend appropriate genetic assessment and counseling for the patients. In this study, multiplex ligation probe dependent amplification (MLPA) analysis was performed on 378 patients with cleft lip and/or palate to characterize rearrangements in patients suspected of 22q11.2 microduplication and microdeletion syndromes. Of 378 cases, 15 were diagnosed with a microdeletion with various sizes and 3 with duplications. For the first time in this study an atypical 0.6 Mb duplication is reported. Illustration of the phenotypes associated with the microduplications increases the knowledge of phenotypes reported in the literature

Negative cross resistance in atrazine resistant junglerice (Echinochloa colona (L.) Link) populations in sugarcane (Saccharum officinarum L.) fields

Introduction Negative and positive cross-resistance to other herbicides have been found in triazine-resistant biotypes. Thus, negative cross-resistance has been reported to bentazone for Brassica napus L. and A. retroflexus L., and to pyridate for B. napus and Epilobium ciliatum Raf.. In contrast, positive cross-resistance to bentazone has been found in Chenopodium album L. and Solanum nigrum L.. Negative cross-resistance, i.e., herbicide-resistant plants being more sensitive to herbicides than susceptible plants, has been documented in several triazine-resistant weed biotypes. The goal of this study was to search for herbicides that exert negative cross-resistance at the whole-plant level for control of atrazine-resistant populations of E. colona. Materials and Methods Suspected resistant and susceptible seeds of junglerice to atrazine were collected from sugarcane fields and adjacent areas of Karun Agro-Industry Inc., Shushtar, southwestern of Iran in 2014 - 2015 growing season. These populations were named according to their population status and abbreviated as follows: R1, R2, R3 and S (susceptible population). All the collected seeds (R- and S-populations) were stored at room temperature (25 oC). Herbicides were tebuthiuron, linuron, diuron and diuron + hexazinone. For the PRE application, 10 seeds were planted in 500-ml plastic pots containing clay:sand mix, and herbicides were applied using an flood nozzle and back sprayer calibrated to deliver 350 L ha-1 to the suspected resistant and susceptible junglerice biotypes 1 d after sowing. Plants were thinned to 4 plants per pot. The aboveground biomass was harvested 28 DAT, dried at 75 °C for 48 h, and weighed. The aboveground biomass data were expressed as a percentage of the untreated control. The experiment was a completely randomized block design with four replications and was repeated. The data were analyzed using a nonlinear regression model and R software (drc add on packeges), the three and four parameters log-logistic equation was fitted to the data to describe the responses of the populations to herbicides: susceptible population was estimated as an index in order to compare the resistance levels of the tested populations. Results and Discussion The result of screening test showed that 3 populations were resistant to atrazine. The results of dose-response assay using atrazine revealed that resistance factor of R1, R2 and R3 populations were 12.26, 6.59 and 3.75 based on wet weight (% of control) and 5.37, 4.22 and 4.71 based on number of survival plants (% of control), respectively. The ED50 values of the R1, R2 and R3 populations were 36.85, 73.01 and 41.71 g ai ha-1 compared with 44.42 g ai ha-1 of the S-populaton of tebuthiuron. The Rf of the R1, R2 and R3 populations were 0.80, 1.53 and 0.72 of tebuthiuron, respectively. The ED50 values of the R1, R2 and R3 populations were 92.76, 107.73 and 106.84 g ai ha-1 compared with 152.51 g ai ha-1 of the S-populaton of linuron. The Rf of the R1, R2 and R3 populations were 1, 1.89 and 3.26 of tebuthiuron, respectively. The ED50 values of the R1, R2 and R3 populations were 74.21, 95.25 and 69.80 g ai ha-1 compared with 79.03 g ai ha-1 of the S-populaton of diuron. The Rf of the R1, R2 and R3 populations were 0.97, 0.85 and 1.18 of diuron, respectively. The ED50 values of the R1, R2 and R3 populations were 62.11, 49.48 and 54.30 g ai ha-1 compared with 88.72 g ai ha-1 of the S-populaton of diuron+hexazinone. The Rf of the R1, R2 and R3 populations were 0.62, 0.68 and 0.52 of diuron+hexazinone, respectively. The results showed that negative cross resistance to tebuthiuron, linuron, diuron and diuron + hexazinone. Resistant biotypes, also, showed the highest negative cross resistance to diuron+hexazinone. Conclusions Some herbicides that inhibit photosystem II bind more efficiently to the mutant triazine binding domain than to the wild (susceptible) type. Triazine-resistant weeds frequently show negative cross-resistance to other photosystem-II inhibitors, such as bentazon and pyridate; triazine-resistant weeds can also exhibit negative cross-resistance to herbicides that do not affect photosystem II. Negative cross resistance may be the major reason that atrazine resistance did not evolve where herbicide mixtures were used, when the mixed herbicide (usually a non-PS II inhibiting acetanilide) also controlled triazine-sensitive weeds. The value of negative cross-resistance linked with the general lack of fitness of almost all triazine-resistant weeds may be greater than we measured herein. The competition exerted by a crop in the field may further accentuate and exacerbate the lack of fitness and further lower the RI

Identification and characterization of the channel-forming protein in the cell wall of Corynebacterium amycolatum

Soltan Mohammadi N, Mafakheri S, Abdali N, Bárcena-Uribarri I, Tauch A, Benz R. Identification and characterization of the channel-forming protein in the cell wall of Corynebacterium amycolatum. Biochimica et biophysica acta. 2013;1828(11):2574-2582.The mycolic-acid layer of certain gram-positive bacteria, the mycolata, represents an additional permeability barrier for the permeation of small water-soluble solutes. Consequently, it was shown in recent years that the mycolic acid layer of individual bacteria of the group mycolata contains pores, called porins, for the passage of hydrophilic solutes. Corynebacterium amycolatum, a pathogenic Corynebacterium species, belongs to the Corynebacteriaceae family but it lacks corynomycolic acids in its cell wall. Despite the absence of corynomycolic acids the cell wall of C. amycolatum contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of C. amycolatum. Based on partial sequencing of the protein responsible for channel formation derived from C. amycolatum ATCC 49368 we were able to identify the gene coram0001_1986 within the known genome sequence of C. amycolatum SK46 that codes for the cell wall channel. The corresponding gene of C. amycolatum ATCC 49368 was cloned into the plasmid pXHis for its expression in Corynebacterium glutamicum ∆porA∆porH. Biophysical characterization of the purified protein (PorAcoram) suggested that coram0001_1986 is indeed the gene coding for the pore-forming protein PorAcoram in C. amycolatum ATCC 49368. The protein belongs to the DUF (Domains of Unknown Function) 3068 superfamily of proteins, mainly found in bacteria from the family Corynebacteriaceae. The nearest relative to PorAcoram within this family is an ORF which codes for PorAcres, which was also recognized in reconstitution experiments as a channel-forming protein in Corynebacterium resistens

Identification of Proximal and Distal 22q11.2 Microduplications among Patients with Cleft Lip and/or Palate: A Novel Inherited Atypical 0.6 Mb Duplication

Misalignments of low-copy repeats (LCRs) located in chromosome 22, particularly band 22q11.2, predispose to rearrangements. A variety of phenotypic features are associated with 22q11.2 microduplication syndrome which makes it challenging for the genetic counselors to recommend appropriate genetic assessment and counseling for the patients. In this study, multiplex ligation probe dependent amplification (MLPA) analysis was performed on 378 patients with cleft lip and/or palate to characterize rearrangements in patients suspected of 22q11.2 microduplication and microdeletion syndromes. Of 378 cases, 15 were diagnosed with a microdeletion with various sizes and 3 with duplications. For the first time in this study an atypical 0.6 Mb duplication is reported. Illustration of the phenotypes associated with the microduplications increases the knowledge of phenotypes reported in the literature

Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long PorACj, Which Forms a Homooligomeric and Anion-Selective Cell Wall Channel

Abdali N, Barth E, Norouzy A, et al. Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long PorACj, Which Forms a Homooligomeric and Anion-Selective Cell Wall Channel. PloS one. 2013;8(10): e75651.Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed

Prevalence of 22q11.2 microdeletion syndrome in Iranian patients with cleft palate

Background: 22q11.2 microdeletion syndrome is the most common multiple genetic disorder associated with learning disabilities, developmental delays, immune deficiency, hypocalcemia, and cleft palate. Finding some valid criteria for screening of 22q11.2 deletion syndromes in infants would be very helpful in early diagnosis and treatment. Materials and Methods: Since 69% of individuals with 22q11.2 deletion have a palatal abnormality, we studied the prevalence of 22q11.2 deletion syndrome in 378 Iranian patients during a 5-year period, including 291 patients affected with cleft palate only without cleft lip (CPO) and 87 patients affected with velopharyngeal incompetence (VPI) and/or submucous cleft palate (SMCP). DNA copy number was analyzed with multiplex ligation-dependent probe amplification (MLPA) technique. Results: In our study, 15/378 (3.97%) patients with palatal anomalies showed 22q11.2 deletion. Interestingly, this prevalence between syndromic patients was 15/104 (14.42%). Conclusion: It seems that SMCP or VPI, in addition to one or more another features of 22q11.2 deletions, especially developmental delay, may be good criteria for molecular investigation of 22q11.2 region

Spatial Planning, Urban Governance and the Economic Context: The Case of ‘Mehr’ Housing Plan, Iran

With the increasing concentration of population and economic activities in metropolitan regions, dwelling shortages and housing quality have become critical issues in urban management. Town plans considering social, economic, political, and cultural features of local communities have been developed with the aim of supporting housing, especially in emerging economies. In Iran, the ‘Mehr Housing’ Plan has been considered as one of the most relevant strategies for social housing since the 2000s. However, the acceptance of ‘Mehr Housing’ plans at the community scale has been rather low, reflecting the fact that it is a top-down, non-participatory policy. The present study investigates the most important factors affecting social acceptance of ‘Mehr Housing’ plans by interviewing 45 experts through a structured questionnaire that evaluated multiple analyses’ dimensions of housing and urban planning in Iran. Results showed that six dimensions (physical, institutional-managerial, economic, socio-cultural, legal, and locational) had contributed to social dissatisfaction with ‘Mehr Housing’ local initiatives. In particular, socio-cultural and legal dimensions were demonstrated to have a large impact on local communities’ dissatisfaction

Analysis of the putative quaternary structure of PorACj.

<p>(A) Model of the octameric form of the PorACj channel in a lipid bilayer PorACj seen perpendicular to the membrane surface. Top view describes the initial setup of eight straight helices arranged in a circular manner to form a tube with a diameter of about 1.4 nm as derived from the experimental measurements. While the secondary structure is colored in purple, the individual amino-acid side chains are depicted as ball chains and colored according to their electrophysiological nature, i.e., neutral/hydrophobic in grey, polar in green, and charged in red (negative) and blue (positive), respectively. The surrounding bilayer is drawn as a grey surface. (B) Side view of the model of the octameric PorACj channel. After a few tenth of nanoseconds of unbiased molecular dynamics simulations, the helices kink in the central region - forming an hourglass shape - where several short side chains of the amino acids are located. The blue ball-stick side chains represent the lysines in the lower region, which are presumably responsible for the ion selectivity of PorACj and which form some kind of constriction zone.</p

Radii of the anions and relative permeability of PorACj from <i>C. jeikeium</i> in different salt solutions.

<p>The data for the limiting conductivities of the different ions were taken from ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075651#pone.0075651-TonThat1" target="_blank">[66]</a>. The radii of the hydrated anions were calculated using the Stokes equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075651#pone.0075651-Trias3" target="_blank">[67]</a>. The single channel conductance of PorACj for the different salts at 0.1 M was taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075651#pone-0075651-t003" target="_blank">Table 3</a>. The relative permeability of the single anions was calculated by dividing the single-channel conductance of the individual anion by that of 0.1 M KBr. The relative permeability for 0.1 M KBr was set to unity.</p