High Prevalence and Significant Association of ESBL and QNR Genes in Pathogenic Klebsiella pneumoniae Isolates of Patients from Kolkata, India

Pathogenic Klebsiella pneumoniae, resistant to beta-lactam and quinolone drugs, is widely recognized as important bacteria causing array of diseases. The resistance property is obtained by acquisition of plasmid encoded bla_TEM, bla_SHV, bla_CTX-M, QNRA, QNRB and QNRS genes. The aim of this study was to document the prevalence and association of these resistant genes in K. pneumoniae infecting patients in India. Approximately 97 and 76.7 % of the 73 K. pneumoniae isolates showed resistance towards beta-lactam and quinolone drugs respectively. Bla genes were detected in 74 % of K. pneumoniae isolates; with prevalence in the following order: bla_TEM > bla_SHV > bla_CTXM. QNR genes were detected in 67 % samples. Chi-square analysis revealed significant association between presence of bla and qnr genes in our study (P value = 0.000125). Sequence analysis of some bla_TEM, bla_SHV, bla_CTX-M and QNRB PCR products revealed presence of bla_TEM1 (GenBank accession: {"type":"entrez-nucleotide","attrs":{"text":"JN193522","term_id":"343796760","term_text":"JN193522"}}JN193522), bla_TEM116 ({"type":"entrez-nucleotide","attrs":{"text":"JN193523","term_id":"343796762","term_text":"JN193523"}}JN193523 and {"type":"entrez-nucleotide","attrs":{"text":"JN193524","term_id":"343796764","term_text":"JN193524"}}JN193524), bla_SHV11, bla_CTXM72 variants ({"type":"entrez-nucleotide","attrs":{"text":"JF523199","term_id":"329024835","term_text":"JF523199"}}JF523199) and QNRB1 ({"type":"entrez-nucleotide","attrs":{"text":"JN193526","term_id":"343796802","term_text":"JN193526"}}JN193526 and {"type":"entrez-nucleotide","attrs":{"text":"JN193527","term_id":"343796804","term_text":"JN193527"}}JN193527) in our samples.     

Draft Genome Sequence of Xylaria grammica EL000614, a Strain Producing Grammicin,a Potent Nematicidal Compound

An endolichenic fungus, Xylaria grammica strain {"type":"entrez-nucleotide","attrs":{"text":"EL000614","term_id":"124922317"}}EL000614, showed strong nematicidal effects against plant pathogenic nematode, Meloidogyne incognita by producing grammicin. We report genome assembly of X. grammica {"type":"entrez-nucleotide","attrs":{"text":"EL000614","term_id":"124922317"}}EL000614 comprised of 25 scaffolds with a total length of 54.73 Mb, N50 of 4.60 Mb, and 99.8% of BUSCO completeness. GC contents of this genome were 44.02%. Gene families associated with biosynthesis of secondary metabolites or regulatory proteins were identified out of 13,730 gene models predicted.     

RNA-Binding Domain is Necessary for PprM Function in Response to the Extreme Environmental Stress in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans was considered as one of the most radiation-resistant organisms on Earth because of its strong resistance to the damaging factors of both DNA and protein, including ionizing radiation, ultraviolet radiation, oxidants, and desiccation. PprM, as a bacterial cold shock protein homolog, was involved in the radiation resistance and oxidative stress response of D. radiodurans, but its potential mechanisms are poorly expounded. In this study, we found that PprM was highly conserved with the RNA-binding domain in Deinococcus genus through performing phylogenic analysis. Moreover, the paper presents the analysis on the tolerance of environmental stresses both in the wild-type and the pprM/pprM RBD mutant strains, demonstrating that pprM and RNA-binding domain disruptant strain were with higher sensitivity than the wild-type strain to cold stress, mitomycin C, UV radiation, and hydrogen peroxide. In the following step, the recombinant PprM was purified, with the finding that PprM was bound to the 5’-untranslated region of its own mRNA by gel mobility shift assay in vitro. With all these findings taken into consideration, it was suggested that PprM act as a cold shock protein and its RNA-binding domain may be involved in reaction to the extreme environmental stress in D. radiodurans.     

Prevalence,antimicrobial resistance profile,and characterization of multi-drug resistant bacteria from various infected wounds in North Egypt

Multi-drug resistant (MDR) bacteria associated with wounds are extremely escalating. This study aims to survey different wounds in Alexandria hospitals, North Egypt, to explore the prevalence and characteristics of MDR bacteria for future utilization in antibacterial wound dressing designs. Among various bacterial isolates, we determined 22 MDR bacteria could resist different classes of antibiotics. The collected samples exhibited the prevalence of mono-bacterial infections (60%), while 40% included poly-bacterial species due to previous antibiotic administration. Moreover, Gram-negative bacteria showed dominance with a ratio of 63.6%, while Gram-positive bacteria reported 36.4%. Subsequently, the five most virulent bacteria were identified following the molecular approach by 16S rRNA and physiological properties using the VITEK 2 automated system. They were deposited in GenBank as Staphylococcus haemolyticus MST1 ({"type":"entrez-nucleotide","attrs":{"text":"KY550377","term_id":"1304487819","term_text":"KY550377"}}KY550377), Pseudomonas aeruginosa MST2 ({"type":"entrez-nucleotide","attrs":{"text":"KY550378","term_id":"1304487820","term_text":"KY550378"}}KY550378), Klebsiella pneumoniae MST3 ({"type":"entrez-nucleotide","attrs":{"text":"KY550379","term_id":"1304487821","term_text":"KY550379"}}KY550379), Escherichia coli MST4 ({"type":"entrez-nucleotide","attrs":{"text":"KY550380","term_id":"1304487822","term_text":"KY550380"}}KY550380), and Escherichia coli MST5 ({"type":"entrez-nucleotide","attrs":{"text":"KY550381","term_id":"1304487823","term_text":"KY550381"}}KY550381). In terms of isolation source, S. haemolyticus MST1 was isolated from a traumatic wound, while P. aeruginosa MST2 and E. coli MST4 were procured from hernia surgical wounds, and K. pneumoniae MST3 and E. coli MST5 were obtained from diabetic foot ulcers. Antibiotic sensitivity tests exposed that K. pneumoniae MST3, E. coli MST4, and E. coli MST5 are extended-spectrum β-lactamases (ESBLs) bacteria. Moreover, S. haemolyticus MST1 belongs to the methicillin-resistant coagulase-negative staphylococcus (MRCoNS), whereas P. aeruginosa MST2 exhibited resistance to common empirical bactericidal antibiotics. Overall, the study provides new insights into the prevalent MDR bacteria in Egypt for further use as specific models in formulating antibacterial wound dressings.     

Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium

Due to its exemplary resistance to ionising radiation, oxidative stress, desiccation and several DNA damaging agents, Deinococcus radiodurans R1 (DR1) is considered as one of the most appropriate candidates for the bioremediation of the nuclear waste sites. However, the high sensitivity of this bacterium to heavy metals, which are usually preponderant at nuclear waste dump sites, precludes its application for bioremediation. This study deals with the expression two metal binding peptides in DR1 as an attractive strategy for developing metal tolerance in this bacterium. A synthetic gene (EC20) encoding a phytochelatin analogue with twenty repeating units of glutamate and cysteine was constructed by overlap extension and expressed in DR1. The cyanobacterial metallothionein (MT) gene, smtA was cloned for intracellular expression in DR1. Both the genes were expressed under the native groESL promoter. DR1 strain carrying the recombinant EC20 demonstrated 2.5-fold higher tolerance to Cd²⁺ and accumulated 1.21-fold greater Cd²⁺ as opposed to the control while the heterologous expression of MT SmtA in DR1 imparted the transformant superior tolerance to Cd²⁺ amassing 2.5-fold greater Cd²⁺ than DR1 expressing EC20.     

Four mutants of Micrococcus radiodurans defective in the ability to repair DNA damaged by mitomycin-C, two of which have wild-type resistance to ultraviolet radiation

Summary Four genes concerned with the resistance of wild-type Micrococcus radiodurans to the lethal action of mitomycin-C (MTC), mtcA, mtcB, uvsA and uvsB, have been identified by isolating mutants sensitive to MTC.Two strains of M. radiodurans, 302 and 262 carrying mutations in mtcA and mtcB respectively, are between forty and sixty times as sensitive as the wild-type to MTC, only slightly more sensitive than the wild-type to ionizing () radiation and have the same resistance as the wild-type to ultraviolet (u.v.) radiation. Strain 302 can be transformed at a high frequency to wild-type resistance to MTC with DNA from strain 262, and vice versa, indicating that mtcA and mtcB have different genetic locations.Two further strains of M. radiodurans, 303 and 263 having mutations in uvsA and uvsB respectively are only from four to eight times as sensitive as the wild-type to MTC, seven to thirteen times as sensitive to -radiation but between twenty to thirty-three times as sensitive to u.v. radiation. Strain 303 can be transformed with DNA from strain 263, or vice versa, to wild-type resistance to u.v. radiation, implying that uvsA and uvsB also have different genetic locations. M. radiodurans strain 301 which is mutant in both mtcA and uvsA, and strain 261 which is mutant in mtcB and uvsB are twenty to forty times as sensitive as the wild-type to both MTC and u.v. radiation and seven to ten times as sensitive to radiation. Neither mtcA and uvsA nor mtcB and uvsB are closely linked.None of the mutant strains is deficient in recombination, as measured by transformation. The repair of MTC-induced DNA damage in M. radiodurans must be different from that described for Escherichia coli.     

PAK4 confers cisplatin resistance in gastric cancer cells via PI3K/Akt- and MEK/ERK-dependent pathways

CDDP [cisplatin or cis-diamminedichloroplatinum(II)] and CDDP-based combination chemotherapy have been confirmed effective against gastric cancer. However, CDDP efficiency is limited because of development of drug resistance. In this study, we found that PAK4 (p21-activated kinase 4) expression and activity were elevated in gastric cancer cells with acquired CDDP resistance (AGS/CDDP and MKN-45/CDDP) compared with their parental cells. Inhibition of PAK4 or knockdown of PAK4 expression by specific siRNA (small interfering RNA)-sensitized CDDP-resistant cells to CDDP and overcome CDDP resistance. Combination treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 [the inhibitor of PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B or PKB) pathway] or PD98509 {the inhibitor of MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] pathway} with PF-3758309 (the PAK4 inhibitor) resulted in increased CDDP efficacy compared with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or PD98509 alone. However, after the concomitant treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PD98509, PF-3758309 administration exerted no additional enhancement of CDDP cytotoxicity in CDDP-resistant cells. Inhibition of PAK4 by PF-3758309 could significantly suppress MEK/ERK and PI3K/Akt signalling in CDDP-resistant cells. Furthermore, inhibition of PI3K/Akt pathway while not MEK/ERK pathway could inhibit PAK4 activity in these cells. The in vivo results were similar with those of in vitro. In conclusion, these results indicate that PAK4 confers CDDP resistance via the activation of MEK/ERK and PI3K/Akt pathways. PAK4 and PI3K/Akt pathways can reciprocally activate each other. Therefore, PAK4 may be a potential target for overcoming CDDP resistance in gastric cancer.     

Thioredoxin System from Deinococcus radiodurans

This paper describes the cloning, purification, and characterization of thioredoxin (Trx) and thioredoxin reductase (TrxR) and the structure determination of TrxR from the ionizing radiation-tolerant bacterium Deinococcus radiodurans strain R1. The genes from D. radiodurans encoding Trx and TrxR were amplified by PCR, inserted into a pET expression vector, and overexpressed in Escherichia coli. The overexpressed proteins were purified by metal affinity chromatography, and their activity was demonstrated using well-established assays of insulin precipitation (for Trx), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) reduction, and insulin reduction (for TrxR). In addition, the crystal structure of oxidized TrxR was determined at 1.9-Å resolution. The overall structure was found to be very similar to that of E. coli TrxR and homodimeric with both NADPH- and flavin adenine dinucleotide (FAD)-binding domains containing variants of the canonical nucleotide binding fold, the Rossmann fold. The K_m (5.7 μM) of D. radiodurans TrxR for D. radiodurans Trx was determined and is about twofold higher than that of the E. coli thioredoxin system. However, D. radiodurans TrxR has a much lower affinity for E. coli Trx (K_m, 44.4 μM). Subtle differences in the surface charge and shape of the Trx binding site on TrxR may account for the differences in recognition. Because it has been suggested that TrxR from D. radiodurans may have dual cofactor specificity (can utilize both NADH and NADPH), D. radiodurans TrxR was tested for its ability to utilize NADH as well. Our results show that D. radiodurans TrxR can utilize only NADPH for activity.Deinococcus radiodurans is a gram-positive bacterium capable of withstanding exposure to extreme gamma ray and UV radiation, oxidants, and desiccation (6, 10, 26). The mechanism behind the ability of D. radiodurans to survive exposure to extreme conditions has been a subject of intense research (10, 43). Its ability to survive exposure to extreme conditions has been attributed a number of factors, as follows: a high number of genome copies (8), ring-like nucleoid organization (22), high manganese content (8), and a higher ability to scavenge reactive oxygen species (ROS) (43). However, the mechanism responsible for its extremophilic nature is not clearly understood (25).Efforts to understand the mechanism behind the capability of D. radiodurans to tolerate extreme conditions have focused on understanding its ability to prevent or repair genomic damage, because if unrepaired, genomic damage is lethal to the cell (7). The ability of D. radiodurans to repair genomic damage is likely due to its ability to prevent proteome damage, i.e., its ability to maintain sufficient enzymatic activity for genome repair after irradiation. Therefore, genome repair probably plays a bigger role than prevention of genome damage in making D. radiodurans radiation tolerant (7, 8). Indeed, some experimental evidence suggests that efficient DNA repair is solely responsible for the ability of D. radiodurans to withstand ionizing radiation. D. radiodurans DNA sustains the same amount of genome damage at high radiation doses as other bacteria, but unlike other bacteria, its damage is mended within hours (25). However, some recent evidence suggests that it is likely that prevention of DNA damage (reactive oxygen species [ROS] scavenging) supplements DNA repair to make D. radiodurans ionizing radiation tolerant. It is worth noting that only about 20% of radiation-induced damage to the genome is due to the direct effect of irradiation (the rest is due to radiation-induced ROS) and that cellular extracts of D. radiodurans are more effective in scavenging ROS than Escherichia coli extracts when subjected to oxidative stress (43). Moreover, D. radiodurans has higher basal levels of some antioxidant enzymatic systems (catalase and superoxide dismutase), and disruption of superoxide dismutase (sodA) and catalase (katA) genes results in increased sensitivity of D. radiodurans to ionizing radiation. In addition D. radiodurans catalase is more resistant to inhibition by substrate H₂O₂ than bovine or Aspergillus niger catalase (17). Taken together, these experimental results suggest a significant contribution of antioxidant systems to the ability of D. radiodurans to withstand extreme ionizing radiation.While the contribution of some antioxidant enzymatic systems to the extremophilic nature of D. radiodurans has been extensively studied, the role of the thioredoxin system has not been investigated (40, 43). The thioredoxin system is composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and various cellular targets. The system is found in both prokaryotes and eukaryotes, and homologues of both TrxR and Trx have been isolated from many species. Trx proteins are low-molecular-mass proteins (12 kDa) that possess a highly conserved active site motif, WCGPC (27, 41). TrxR is a homodimeric enzyme and is a member of the family of pyridine nucleotide-disulfide oxidoreductase flavoenzymes. Each monomer possesses a flavin adenine dinucleotide (FAD) prosthetic group, a NADPH-binding site, and an active site comprising a redox-active disulfide. There are two distinct forms of this enzyme, as follows: low-molecular-mass TrxR (35 kDa), found in prokaryotes and some eukaryotes, and high-molecular-mass TrxR (55 kDa), found in eukaryotes (41). The two types of TrxR proteins have some differences in structure and mechanism. However, in both cases, reducing equivalents are transferred from NADPH to TrxR, from TrxR to Trx, and finally, from Trx to various cellular proteins (29, 41). Trx targets include proteins which take part in the scavenging of ROS-like thioredoxin-dependent thiol peroxidase (29). The thioredoxin system is thus an important antioxidant enzymatic system.In this study we report the expression, purification, and biochemical characterization of the main components of the D. radiodurans thioredoxin system. In addition, the structural characterization of D. radiodurans TrxR is reported.     

Migratory birds as the vehicle of transmission of multi drug resistant extended spectrum β lactamase producing Escherichia fergusonii,an emerging zoonotic pathogen

The acquisition of multi-drug resistance (MDR) genes by pathogenic bacterial bugs and their dispersal to different food webs has become a silent pandemic. The multiplied use of different antibacterial therapeutics during COVID-19 pandemic has accelerated the process among emerging pathogens. Wild migratory birds play an important role in the spread of MDR pathogens and MDR gene flow due to the consumption of contaminated food and water. Escherichia fergusonii is an emerging pathogen of family Enterobacteriaceae and commonly causes disease in human and animals. The present study focused on the isolation of E. fergusonii from blood, saliva, and intestine of selected migratory birds of the Hazara Division. The sensitivity of isolated strains was assessed against ten different antibiotics. The isolation frequency of E. fergusonii was 69%. In blood samples, a high rate of resistance was observed against ceftriaxone (80%) followed by ampicillin (76%) whereas, in oral and intestinal samples, ceftriaxone resistant strains were 56% and 57% while ampicillin resistance was 49% and 52% respectively. The overall ceftriaxone and ampicillin-resistant cases in all three sample sources were 71% and 65% respectively. In comparison to oral and intestinal samples, high numbers of ceftriaxone-resistant strains were isolated from the blood of mallard while ampicillin-resistant strains were observed in blood samples of cattle egrets. 16S rRNA-based confirmed strains of E. fergusonii were processed for detection of CTX-M and TEM-1 gene through Polymerase chain reaction (PCR) after DNA extraction. Hundred percent ceftriaxone resistant isolates possessed CTX-M and all ampicillin-resistant strains harbored TEM-1 genes. Amplified products were sequenced by using the Sanger sequencing method and the resulted sequences were checked for similarity in the nucleotide Database through the BLAST program. TEM-1 gene showed 99% and the CTX-M gene showed 98% similar sequences in the Database. The 16S rRNA sequence and nucleotide sequences for TEM-1 and CTX-M genes were submitted to Gene Bank with accession numbers {"type":"entrez-nucleotide","attrs":{"text":"LC521304","term_id":"1806500528","term_text":"LC521304"}}LC521304, {"type":"entrez-nucleotide","attrs":{"text":"LC521306","term_id":"1806500530","term_text":"LC521306"}}LC521306, {"type":"entrez-nucleotide","attrs":{"text":"LC521307","term_id":"1806500532","term_text":"LC521307"}}LC521307 respectively. We posit to combat MDR gene flow among the bacterial pathogens across different geographical locations, regular surveillance of new zoonotic pathogens must be conducted.     

Knockout of <Emphasis Type="Italic">pprM</Emphasis> Decreases Resistance to Desiccation and Oxidation in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans has attracted a great interest in the past decades due to its extraordinary resistance to ionizing radiation and highly efficient DNA repair system. Recent studies indicated that pprM is a putative pleiotropic gene in D. radiodurans and plays an important role in radioresistance and antioxidation, but its underlying mechanisms are poorly elucidated. In this study, pprM mutation was generated to investigate resistance to desiccation and oxidative stress. The result showed that the survival of pprM mutant under desiccation was markedly retarded compared to the wild strain from day 7–28. Furthermore, knockout of pprM increases the intercellular accumulation of ROS and the sensibility to H₂O₂ stress in the bacterial growth inhibition assay. The absorbance spectrum experiment for detecting the carotenoid showed that deinoxanthin, a carotenoid that peculiarly exists in Deinococcus, was reduced in the pprM mutant in the pprM mutant. Quantitative real time PCR showed decreased expression of three genes viz. CrtI (DR0861, 50%),CrtB (DR0862, 40%) and CrtO (DR0093, 50%), which are involved in deinoxanthin synthesis, and of Dps (DNA protection during starving) gene (DRB0092) relevant to ion combining and DNA protection in cells. Our results suggest that pprM may affect antioxidative ability of D. radiodurans by regulating the synthesis of deinoxanthin and the concentration of metal ions. This may provide new clues for the treatment of antioxidants.     

PprA,a pleiotropic protein for radioresistance,works through DNA gyrase and shows cellular dynamics during postirradiation recovery in Deinococcus radiodurans

PprA, a pleiotropic protein involved in radioresistance of Deinococcus radiodurans was detected in multiprotein DNA processing complex identified from this bacterium. pprA mutant expressing GFP-PprA could restore its wild type resistance of γ radiation. Under normal conditions, GFP-PprA expressing cells showed PprA localization on both septum trapped nucleoids (STN) and nucleoids located elsewhere (MCN). Cell exposed to 4 kGy γ radiation showed nearly 2 h growth lag and during this growth arrest phase, the majority of the cells had GFP-PprA located on MCN. While in late phase (～120 min) PIR cells, when cells are nearly out of growth arrest, PprA was maximally found with STN. These cells when treated with nalidixic acid showed diffused localization of PprA across the septum. gyrA disruption mutant of D. radiodurans showed growth inhibition, which increased further in gyrA pprA mutant. Interestingly, gyrA mutant showed ～20-fold less resistance to γ radiation as compared to wild type, which did increase further in gyrA pprA mutant. These results suggested that PprA localization undergoes a dynamic change during PIR, and its localization on nucleoid near septum and functional interaction with gyrase A might suggest a mechanism that could explain PprA role in genome segregation possibly through topoisomerase II.     

Genetic organisation, mobility and predicted functions of genes on integrated, mobile genetic elements in sequenced strains of Clostridium difficile

Background

Clostridium difficile is the leading cause of hospital-associated diarrhoea in the US and Europe. Recently the incidence of C. difficile-associated disease has risen dramatically and concomitantly with the emergence of ‘hypervirulent’ strains associated with more severe disease and increased mortality. C. difficile contains numerous mobile genetic elements, resulting in the potential for a highly plastic genome. In the first sequenced strain, 630, there is one proven conjugative transposon (CTn), Tn5397, and six putative CTns (CTn1, CTn2 and CTn4-7), of which, CTn4 and CTn5 were capable of excision. In the second sequenced strain, {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, two further CTns were described.

Results

CTn1, CTn2 CTn4, CTn5 and CTn7 were shown to excise from the genome of strain 630 and transfer to strain CD37. A putative CTn from {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, misleadingly termed a phage island previously, was shown to excise and to contain three putative mobilisable transposons, one of which was capable of excision. In silico probing of C. difficile genome sequences with recombinase gene fragments identified new putative conjugative and mobilisable transposons related to the elements in strains 630 and {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291. CTn5-like elements were described occupying different insertion sites in different strains, CTn1-like elements that have lost the ability to excise in some ribotype 027 strains were described and one strain was shown to contain CTn5-like and CTn7-like elements arranged in tandem. Additionally, using bioinformatics, we updated previous gene annotations and predicted novel functions for the accessory gene products on these new elements.

Conclusions

The genomes of the C. difficile strains examined contain highly related CTns suggesting recent horizontal gene transfer. Several elements were capable of excision and conjugative transfer. The presence of antibiotic resistance genes and genes predicted to promote adaptation to the intestinal environment suggests that CTns play a role in the interaction of C. difficile with its human host.     

Intracellular Copper Accumulation Enhances the Growth of Kineococcus radiotolerans during Chronic Irradiation

The actinobacterium Kineococcus radiotolerans is highly resistant to ionizing radiation, desiccation, and oxidative stress, though the underlying biochemical mechanisms are unknown. The purpose of this study was to explore a possible linkage between the uptake of transition metals and extreme resistance to ionizing radiation and oxidative stress. The effects of six different divalent cationic metals on growth were examined in the absence of ionizing radiation. None of the metals tested were stimulatory, though cobalt was inhibitory to growth. In contrast, copper supplementation dramatically increased colony formation during chronic irradiation. K. radiotolerans exhibited specific uptake and intracellular accumulation of copper, compared to only a weak response to both iron and manganese supplementation. Copper accumulation sensitized cells to hydrogen peroxide. Acute-irradiation-induced DNA damage levels were similar in the copper-loaded culture and the age-synchronized no-copper control culture, though low-molecular-weight DNA was more persistent during postirradiation recovery in the Cu-loaded culture. Still, the estimated times for genome restoration differed by only 2 h between treatments. While we cannot discount the possibility that copper fulfills an unexpectedly important biochemical role in a low-radioactivity environment, K. radiotolerans has a high capacity for intracellular copper sequestration and presumably efficiently coordinated oxidative stress defenses and detoxification systems, which confers cross-protection from the damaging effects of ionizing radiation.     

LeoA,B and C from Enterotoxigenic Escherichia coli (ETEC) Are Bacterial Dynamins

Escherichia coli (ETEC) strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 contains a GTPase virulence factor, LeoA, which is encoded on a pathogenicity island and has been shown to enhance toxin release, potentially through vesicle secretion. By sequence comparisons and X-ray structure determination we now identify LeoA as a bacterial dynamin-like protein (DLP). Proteins of the dynamin family remodel membranes and were once thought to be restricted to eukaryotes. In ETEC {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 LeoA localises to the periplasm where it forms a punctate localisation pattern. Bioinformatic analyses of leoA and the two upstream genes leoB and leoC suggest that LeoA works in concert with a second dynamin-like protein, made up of LeoB and LeoC. Disruption of the leoAB genes leads to a reduction in secretion of periplasmic Tat-GFP and outer membrane OmpA. Our data suggest a role for LeoABC dynamin-like proteins in potentiating virulence through membrane vesicle associated toxin secretion.     

<Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> RecX and <Emphasis Type="Italic">Escherichia coli</Emphasis> RecX proteins are capable to replace each other in vivo and in vitro

A plasmid carrying the Deinococcus radiodurans recX gene under the control of a lactose promoter decreases the Escherichia coli cell resistance to UV irradiation and γ irradiation and also influences the conjugational recombination process. The D. radiodurans RecX protein functions in the Escherichia coli cells similarly to the E. coli RecX protein. Isolated and purified D. radiodurans RecX and E. coli RecX proteins are able to replace each other interacting with the E. coli RecA and D. radiodurans RecA proteins in vitro. Data obtained demonstrated that regulatory interaction of RecA and RecX proteins preserves a high degree of conservatism despite all the differences in the recombination reparation system between E. coli and D. radiodurans.     

Classifying Integrated Signature Molecules in Macrophages of Rheumatoid Arthritis,Osteoarthritis, and Periodontal Disease: An Omics-Based Study

Rheumatoid arthritis (RA), osteoarthritis (OA), and periodontal disease (PD) are chronic inflammatory diseases that are globally prevalent, and pose a public health concern. The search for a potential mechanism linking PD to RA and OA continues, as it could play a significant role in disease prevention and treatment. Recent studies have linked RA, OA, and PD to Porphyromonas gingivalis (PG), a periodontal bacterium, through a similar dysregulation in an inflammatory mechanism. This study aimed to identify potential gene signatures that could assist in early diagnosis as well as gain insight into the molecular mechanisms of these diseases. The expression data sets with the series IDs {"type":"entrez-geo","attrs":{"text":"GSE97779","term_id":"97779"}}GSE97779, {"type":"entrez-geo","attrs":{"text":"GSE123492","term_id":"123492"}}GSE123492, and {"type":"entrez-geo","attrs":{"text":"GSE24897","term_id":"24897"}}GSE24897 for macrophages of RA, OA synovium, and PG stimulated macrophages (PG-SM), respectively, were retrieved and screened for differentially expressed genes (DEGs). The 72 common DEGs among RA, OA, and PG-SM were further subjected to gene–gene correlation analysis. A GeneMANIA interaction network of the 47 highly correlated DEGs comprises 53 nodes and 271 edges. Network centrality analysis identified 15 hub genes, 6 of which are DEGs (API5, ATE1, CCNG1, EHD1, RIN2, and STK39). Additionally, two significantly up-regulated non-hub genes (IER3 and RGS16) showed interactions with hub genes. Functional enrichment analysis of the genes showed that “apoptotic regulation” and “inflammasomes” were among the major pathways. These eight genes can serve as important signatures/targets, and provide new insights into the molecular mechanism of PG-induced RA, OA, and PD.     

A Genomewide Overexpression Screen Identifies Genes Involved in the Phosphatidylinositol 3-Kinase Pathway in the Human Protozoan Parasite Entamoeba histolytica

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen.     

Occurrence and Molecular Identification of Anisakis Dujardin, 1845 from Marine Fish in Southern Makassar Strait,Indonesia

Anisakis spp. (Nematoda: Anisakidae) parasitize a wide range of marine animals, mammals serving as the definitive host and different fish species as intermediate or paratenic hosts. In this study, 18 fish species were investigated for Anisakis infection. Katsuwonus pelamis, Euthynnus affinis, Caranx sp., and Auxis thazard were infected with high prevalence of Anisakis type I, while Cephalopholis cyanostigma and Rastrelliger kanagurta revealed low prevalence. The mean intensity of Anisakis larvae in K. pelamis and A. thazard was 49.7 and 5.6, respectively. A total of 73 Anisakis type I larvae collected from K. pelamis and A. thazard were all identified as Anisakis typica by PCR-RFLP analysis. Five specimens of Anisakis from K. pelamis and 15 specimens from A. thazard were sequenced using ITS1-5.8S-ITS2 region and 6 specimens from A. thazard and 4 specimens from K. pelamis were sequenced in mtDNA cox2 region. Alignments of the samples in the ITS region showed 2 patterns of nucleotides. The first pattern (genotype) of Anisakis from A. thazard had 100% similarity with adult A. typica from dolphins from USA, whereas the second genotype from A. thazard and K. pelamis had 4 base pairs different in ITS1 region with adult A. typica from USA. In the mtDNA cox2 regions, Anisakis type I specimens from A. thazard and K. pelamis showed similarity range from 94% to 99% with A. typica {"type":"entrez-nucleotide","attrs":{"text":"AB517571","term_id":"283379497","term_text":"AB517571"}}AB517571/{"type":"entrez-nucleotide","attrs":{"text":"DQ116427","term_id":"74229655","term_text":"DQ116427"}}DQ116427. The difference of 4 bp nucleotides in ITS1 regions and divergence into 2 subgroups in mtDNA cox2 indicating the existence of A. typica sibling species in the Makassar Strait.