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Staphylococcus aureus (S. aureus) is a widespread foodborne pathogen, causing a serious threat to human health and public safety. Thus, it is significant for rapid S. aureus determination. Herein, a visual and smartphone-integrated aptasensor for intact S. aureus was fabricated based on the Exo III-assisted recycling and hemin/G-quadruplex DNAzymes for signal amplification. Different concentrations of S. aureus caused the difference in releasing different amounts of DNAzymes via Exo III-assisted recycling reaction. These DNAzymes catalyzed the oxidation of 2,2′-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS2-) to colored ABTS•−, which were respectively analyzed by smartphone and UV-Vis spectrophotometer for quantification. This assay exhibited good analytical performance with a detection limit of 32 CFU·mL−1 and a wide linear range. It also possesses the ability in complex sample matrices detection, low cost, and portability with the employment of a smartphone, making it a rapid, simple, and point-of-care test (POCT) method with great potential in food safety control. • A smartphone-integrated dual-readout aptasensor for Staphylococcus aureus was constructed. • This method showed a good analytical performance and met the POCT requirement for application in food safety control. • A versatile platform for simple and intact detection of biomolecules was developed. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus) is a widespread foodborne pathogen, causing a serious threat to human health and public safety. Thus, it is significant for rapid S. aureus determination. Herein, a visual and smartphone-integrated aptasensor for intact S. aureus was fabricated based on the Exo III-assisted recycling and hemin/G-quadruplex DNAzymes for signal amplification. Different concentrations of S. aureus caused the difference in releasing different amounts of DNAzymes via Exo III-assisted recycling reaction. These DNAzymes catalyzed the oxidation of 2,2′-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS2-) to colored ABTS•−, which were respectively analyzed by smartphone and UV-Vis spectrophotometer for quantification. This assay exhibited good analytical performance with a detection limit of 32 CFU·mL−1 and a wide linear range. It also possesses the ability in complex sample matrices detection, low cost, and portability with the employment of a smartphone, making it a rapid, simple, and point-of-care test (POCT) method with great potential in food safety control. • A smartphone-integrated dual-readout aptasensor for Staphylococcus aureus was constructed. • This method showed a good analytical performance and met the POCT requirement for application in food safety control. • A versatile platform for simple and intact detection of biomolecules was developed. [ABSTRACT FROM AUTHOR]

In order to improve the Internet coverage ratio and provide connectivity guarantee, based on sensor opportunistic coverage mechanism and cooperative cloud service, we proposed the coverage connectivity guarantee protocol for mobile Internet. In this scheme, based on the opportunistic covering rules, the network coverage algorithm of high reliability and real-time security was achieved by using the opportunity of sensor nodes and the Internet mobile node. Then, the cloud service business support platform is created based on the Internet application service management capabilities and wireless sensor network communication service capabilities, which is the architecture of the cloud support layer. The cooperative cloud service aware model was proposed. Finally, we proposed the mobile Internet coverage connectivity guarantee protocol. The results of experiments demonstrate that the proposed algorithm has excellent performance, in terms of the security of the Internet and the stability, as well as coverage connectivity ability. [ABSTRACT FROM AUTHOR]

Abstract: The ColR–ColS two-component signal transduction system was originally characterized as a regulatory system involved in the capacity of root-colonizing biocontrol bacterium Pseudomonas fluorescens to colonize plant roots. There are three pairs of putative colR–colS two-component regulatory systems annotated in the phytopathogen Xanthomonas campestris pathovar campestris. Mutational studies revealed that one of them, named colRXC1049 and colSXC1050 , is a global regulatory system involved in various cellular processes, including virulence, hypersensitive response and stress tolerance. Growth rate determination showed that, although the colRXC1049 and colSXC1050 mutants are not auxotrophic, colRXC1049 and colSXC1050 are required for the pathogen to proliferate well in standard media and host plants. Assays of β-glucuronidase activities of plasmid-driven promoter-gusA reporters and/or semi-quantitative RT-PCR demonstrated that colRXC1049 and colSXC1050 positively regulate expression of hrpC and hrpE operons, and that expression of colRXC1049 and colSXC1050 is not controlled by key hrp regulators HrpG and HrpX. [Copyright &y& Elsevier]

Abstract: XopN was originally identified from Xanthomonas campestris pv. vesicatoria as an effector translocated into plant cells via the type III secretion system (T3SS), and is required for pathogenicity. We report here that the xopN homologue in the X. campestris pv. campestris genome, named xopXccN, also encodes a T3SS effector and is required for full virulence. We further demonstrate that expression of xopXccN is positively regulated by the key hrp (hypersensitive response and pathogenicity) regulators HrpG and HrpX. [Copyright &y& Elsevier]

In this work, a sensitive electrochemical method for bleomycin (BLM) determination was reported on the basis of BLM-mediated activation of Zn2+-dependent DNAzyme and the adsorption of signal probes by a metal-organic framework (MOF) modified electrode. Two hairpin DNAs were employed in this protocol, one (HP1) for BLM recognition and one (HP2) for amplified signal output. The presence of BLM and Fe2+ caused the formation of BLM-Fe (II) complex to cleave HP1, releasing DNAzyme fragments, which could further hybridize with substrate HP2 to form a partial double-stranded DNA duplex and enable the activation of Zn2+-dependent DNAzyme with the coexistence of Zn2+. The Zn2+-dependent DNAzyme catalyzed the cyclic cleavage of magnetic beads (MB)-immobilized HP2 to release massive DNA fragments with a Fc-labeled- terminal, which could be used for BLM quantification through electrochemical measurement after their adsorption on a MOF modified electrode. Attributing to the high catalytic efficiency of DNAzyme and excellent electrochemical performance of MOF modified electrode, our method revealed an impressive limit of detection as low as 4 pM BLM with a linear range of 5–2000 pM. Besides, the easy synthesis of MOF without further modification and the easy way of adsorption for signal achievement facilitated the operation process. In virtue of the high sensitivity, selectivity and the simple-to-implement features, this method is believed to hold a great promising application for BLM determination in biomedical and clinical study. • Target-mediated activation of Zn2+-dependent DNAzyme was utilized for amplified bleomycin determination. • The MOF-based electrochemical signal was creatively and easily achieved without complicated modification. • This method revealed a low detection limit, a wide linear range and the ability for complex sample analysis. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS), a multifunctional protein secretion device, plays very important roles in bacterial killing and/or virulence to eukaryotic cells. Although T6SS genes have been found in many Xanthomonas species, the biological function of T6SSs has not been elucidated in most xanthomonads. In this study, we identified two phylogenetically distinct T6SS clusters, T6SS1 and T6SS2, in a newly sequenced Chinese strain GX01 of Xanthomonas oryzea pv. oryzicola (Xoc) which causes bacterial leaf streak (BLS) of rice (Oryza sativa L.). Mutational assays demonstrated that T6SS1 and T6SS2 are not required for the virulence of Xoc GX01 on rice. Nevertheless, we found that T6SS2, but not T6SS1, played an important role in bacterial killing. Transcription and secretion analysis revealed that hcp2 gene is actively expressed and that Hcp2 protein is secreted via T6SS. Moreover, several candidate T6SS effectors were predicted by bioinformatics analysis that might play a role in the antibacterial activity of Xoc. This is the first report to investigate the type VI secretion system in Xanthomonas oryzae. We speculate that Xoc T6SS2 might play an important role in inter-bacterial competition, allowing this plant pathogen to gain niche advantage by killing other bacteria. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pv. oryzicola (Xoc) depends on its type III secretion system (T3SS) to translocate type III secreted effectors (T3SEs), including transcription activator-like effectors (TALEs) and non-transcription activator-like effectors (non-TALEs), into host cells. T3SEs can promote the colonization of Xoc and contribute to virulence by manipulating host cell physiology. We annotated 25 genes encoding non-TALEs in Xoc strain GX01, an isolate from Guangxi in the South China's rice growing region. Through systematic mutagenesis of non-TALEs, we found that xopN , the virulence contribution of which was previously unknown for Xoc , significantly contributes to the virulence of Xoc GX01, as does avrBs2. [ABSTRACT FROM AUTHOR]

Xanthomonas is a well-studied genus of bacterial plant pathogens whose members cause a variety of diseases in economically important crops worldwide. Genomic and functional studies of these phytopathogens have provided significant understanding of microbial-host interactions, bacterial virulence and host adaptation mechanisms including microbial ecology and epidemiology. In addition, several strains of Xanthomonas are important as producers of the extracellular polysaccharide, xanthan, used in the food and pharmaceutical industries. This polymer has also been implicated in several phases of the bacterial disease cycle. In this review, we summarise the current knowledge on the infection strategies and regulatory networks controlling virulence and adaptation mechanisms from Xanthomonas species and discuss the novel opportunities that this body of work has provided for disease control and plant health. [ABSTRACT FROM AUTHOR]

• A panel of PEDV structural and nonstructural proteins were expressed. • These PEDV antigens were tested for reactivity with sera from PEDV-infected pigs. • 851 porcine sera were analyzed by ELISA with antigens showing immune-reactivity. • Pattern of neutralizing antibody was more similar to that of IgA in weaning piglets. • ORF3C and E in addition to S1 can be used as diagnostic markers for serologic detection. Given the highly contagious and acute nature of porcine epidemic diarrhea (PED), especially in piglets, there is an urgent need for the development of rapid and sensitive diagnostic assays. The diagnostic potentials of specific porcine epidemic diarrhea virus (PEDV) accessory and nonstructural proteins, if any, have not yet been investigated. In order to determine and compare which of the viral proteins may be useful as diagnostic antigens, whole virus (WV) particles and a panel of structural and nonstructural PEDV proteins [spike subunit 1 (S1), the C-terminal part of ORF3 (ORF3C), envelope (E), nonstructural protein 1 (Nsp1), Nsp2, Ac (acidic domain of Nsp3), and ADRP (ADP-ribose-1-monophosphatase domain of Nsp3), expressed individually in bacterial and/or mammalian cells] were tested for reactivity with sera from PEDV-infected pigs by ELISA and/or western blot analysis. According to western blots, serum antibody interactions with the S1 protein were relatively more sensitive and specific than ORF3C, E and Ac. Furthermore, a total of 851 serum samples from diarrheal pigs of different ages were analyzed by ELISA, with most showing immune-reactivity towards the WV, S1, ORF3C, and E proteins. The earliest IgG antibody response was observed in the one-week-old piglets, with similar antibody ontogeny and patterns of seroconversion for S1, ORF3C, E, and WV antigens. In addition, the pattern of neutralizing antibody was more similar to that of IgA in weaning piglets after PEDV infection. Collectively, these data provide more reliable information on the host immune response to different viral proteins, which will be useful for development of novel serological assays and for design of vaccines that better stimulate protective immunity. [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Xanthomonas oryzae pv. oryzicola (Xoc) is the causal agent of rice bacterial leaf streak (BLS), one of the most destructive diseases of rice (Oryza sativa L.) that is the important staple crop. Xoc can invade host leaves via stomata and wounds and its type three secretion system (T3SS) is pivotal to its pathogenic lifestyle. In this study, using a novel dual RNA-seq approach, we examined transcriptomes of rice and Xoc in samples inoculated with wild type Xoc GX01 and its T3SS defective strain (T3SD), to investigate the global transcriptional changes in both organisms. Compared with T3SD strain, rice inoculated with wild type Xoc GX01 resulted in significant expression changes of a series of plant defence related genes, including ones altered in plant signalling pathway, and downregulated in phenylalanine metabolism, flavonoid and momilactone biosynthesis, suggesting repression of plant defence response and reduction in both callose deposition and phytoalexin accumulation. Also, some known transcription activator-like effector (TALE) targets were induced by Xoc GX01, e.g. OsSultr3;6 which contributes to rice susceptibility. Some cell elongation related genes, including several expansin genes, were induced by GX01 too, suggesting that Xoc may exploit this pathway to weaken cell wall strength, beneficial for bacterial infection. On the other hand, compared with wild type, the T3SD strain transcriptome in planta was characterized by downregulation of ATP, protein and polysaccharide synthesis, and upregulation of antioxidation and detoxification related genes, revealing that T3SD strain faced serious starvation and oxidation stresses in planta without a functional T3SS. In addition, comparative global transcript profiles of Xoc in planta and in medium revealed an upregulation of virulence factor synthesis and secretion in planta in favour of bacterial infection. Collectively, this study provides a comprehensive representation of cross talk between the host and bacterial pathogen, revealing insights into the Xoc-rice pathogenic dynamic and reveals novel strategies exploited by this important pathogen to cause disease. [ABSTRACT FROM AUTHOR]

A correction to the article "Cyclic di-GMP Signalling in the Virulence and Environmental Adaptation of Xanthomonas Campestris," by Robert P. Ryan and colleagues is presented.

A number of species of bacteria from the genus B urkholderia have been shown to be causal agents of diseases of rice. These diseases, caused by B urkholderia glumae, B . gladioli and B . plantarii, are becoming increasingly common across the globe. This is particularly so for B . glumae, whose ability to grow at elevated temperatures suggests that it may become a prevalent problem in an era of global warming. Despite the increasing threat to rice, relatively little is known about the virulence mechanisms employed by these pathogens. Work over the last 5 years has provided an increasing insight into these factors and their control by environmental and other cues. In addition, the determination of a number of genome sequences has allowed bioinformatic predictions of further possible mechanisms, which can now be investigated experimentally. Here, we review recent advances in the understanding of virulence of B urkholderia to rice, to include discussion of the roles of toxins, type II secreted enzymes, type III secreted effectors and motility as well as their regulation by quorum sensing, two-component systems and cyclic di- GMP signalling. Finally, we consider a number of approaches for the control of bacterial virulence through the modulation of quorum sensing and toxin degradation. [ABSTRACT FROM AUTHOR]

Xanthomonas TALE transcriptional activators act as virulence or avirulence factors by activating host disease susceptibility or resistance genes. Their specificity is determined by a tandem repeat domain. Some Xanthomonas pathogens contain 10-30 TALEs per strain. Although TALEs play critical roles in pathogenesis, their studies have so far been limited to a few examples, due to their highly repetitive gene structure and extreme similarity among different members, which constrict sequencing and assembling. To facilitate TALE studies, we developed an efficient and rapid pipeline for genome-wide cloning of tal genes as many as possible from a strain. Here, we report the pipeline and its use to identify all 18 tal genes from a newly isolated strain of the rice pathogen Xathomonas oryzae. Target prediction revealed a number of potential rice targets including several notable genes such as genes encoding SWEET, WRKY, Hen1, and BAK1 proteins, which provide candidates for further experimental functional analysis of the TALEs. [ABSTRACT FROM AUTHOR]

The bacterial phytopathogen X anthomonas campestris pv. campestris ( Xcc) relies on the hrp (hypersensitive response and pathogenicity) genes to cause disease and induce hypersensitive response ( HR). The hrp genes of bacterial phytopathogens are divided into two groups. Xcc hrp genes belong to group II. It has long been known that the group II hrp genes are activated by an AraC-type transcriptional regulator whose expression is controlled by a two-component system ( TCS) response regulator (named HrpG in Xcc). However, no cognate sensor kinase has yet been identified. Here, we present evidence showing that the Xcc open-reading frame XC _3670 encodes a TCS sensor kinase (named HpaS). Mutation of hpaS almost completely abolished the HR induction and virulence. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacted with HrpG. Phos-tag™ SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of HrpG in vivo. These data suggest that HpaS and HrpG are most likely to form a TCS. We also showed that XC _3669 (named hpaR2), which is adjacent to hpaS and encodes a putative TCS response regulator, is required for full virulence but not HR induction. HpaR2 also physically interacted with HpaS, suggesting that HpaS may also form another TCS with HpaR2. [ABSTRACT FROM AUTHOR]

Abstract: Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease in cruciferous plants worldwide. Although the complete genomes of several Xcc strains have been determined, the gene expression and regulation mechanisms in this pathogen are far from clear. In this work, transcriptome profiling of Xcc 8004 grown in MMX medium (minimal medium for Xanthomonas campestris) and NYG medium (peptone yeast glycerol medium) were investigated by RNA-Seq. Using the Illumina HiSeq 2000 platform, a total of 26,514,630 reads (90 nt in average) were generated, of which 15,708,478 reads mapped uniquely to coding regions of Xcc 8004 genome. Of the 4273 annotated protein-coding genes of Xcc 8004, 629 were found differentially expressed in Xcc grown in MMX and NYG. Of the differentially expressed genes, 495 were up-regulated and 134 were down-regulated in MMX. The MMX-induced genes are mainly involved in amino acid metabolism, transport systems, atypical condition adaptation and pathogenicity, especially the type III secretion system, while the MMX-repressed genes are mainly involved in chemotaxis and degradation of small molecules. The global transcriptome analyzes of Xcc 8004 grown in MMX and NYG might facilitate the gene functional characterization of this phytopathogenic bacterium. [Copyright &y& Elsevier]

Abstract: The mosquito is a very important vector involved in the worldwide transmission of disease-causing viruses and parasites. Controlling the mosquito population remains one of the best means for preventing the serious infectious diseases of malaria, yellow fever, dengue, filariasis and so on and there has been an increasing interest in developing biopesticides as a useful substitute to chemical insecticides. As a result, Bacillus thuringiensis subsp. israelensis (Bti) has been extensively used due to its specificity and high toxicity to a variety of mosquito larvae. However it is prudent to seek alternatives to Bti with alternative spectra of mosquitocidal activity or that are able to overcome any resistance that might develop against Bti. The Bt S2160-1 strain was isolated from soil samples collected from Southern China and found to have a comparable mosquitocidal activity to Bti. However there were significant differences in terms of their plasmid profiles, crystal proteins produced and cry gene complement. A PCR-restriction fragment length polymorphism identification system was developed and used in order to identify novel cry-type genes and four such genes (cry30Ea, cry30Ga, cry50Ba and cry54Ba) were identified in Bt S2160-1. In conclusion, Bt S2160-1 has been identified as a potential alternative to Bti, which could be used for the control of mosquito populations in order to reduce the incidence of mosquito-borne diseases. [Copyright &y& Elsevier]

Mip (macrophage infectivity potentiator) and Mip-like proteins have been demonstrated to be involved in virulence of several animal pathogens, but as yet none of their native bacterial targets has been identified. Our previous work demonstrated that the Mip-like protein found in the plant pathogen X anthomonas campestris pv. campestris ( Xcc) (hereafter called MipXcc) is also involved in virulence. Inactivation of the mipXcc gene leads to a significant reduction in exopolysaccharide production and extracellular protease activity via an unknown mechanism. The Xcc genome encodes six extracellular proteases, all of which are secreted via the type II secretion system. The serine protease PrtA makes the largest contribution to Xcc's total extracellular proteolytic activity. In this study, Western blotting analysis demonstrated that MipXcc was located in the periplasm. Bacterial two-hybrid and far-Western analysis indicated that MipXcc interacted with PrtA directly. Purified MipXcc was found to be able to rescue the protease activity of periplasmic proteins extracted from the mipXcc mutant. These findings show that MipXcc plays a role in the maturation of PrtA, which is the novel native target for at least one Mip or Mip-like protein. [ABSTRACT FROM AUTHOR]

Cyclic di-GMP is a second messenger with a role in regulation of a range of cellular functions in diverse bacteria including the virulence of pathogens. Cellular levels of cyclic di-GMP are controlled through synthesis, catalysed by the GGDEF protein domain, and degradation by EAL or HD-GYP domains. Here we report a comprehensive study of cyclic di-GMP signalling in bacterial disease in which we examine the contribution of all proteins with GGDEF, EAL or HD-GYP domains to virulence and virulence factor production in the phytopathogen Xanthomonas campestris pathovar campestris ( Xcc). Genes with significant roles in virulence to plants included those encoding proteins whose probable function is in cyclic-di-GMP synthesis as well as others (including the HD-GYP domain regulator RpfG) implicated in cyclic di-GMP degradation. Furthermore, RpfG controlled expression of a subset of these genes. A partially overlapping set of elements controlled the production of virulence factors in vitro. Other GGDEF-EAL domain proteins had no effect on virulence factor synthesis but did influence motility. These findings indicate the existence of a regulatory network that may allow Xcc to integrate information from diverse environmental inputs to modulate virulence factor synthesis as well as of cyclic di-GMP signalling systems dedicated to other specific tasks. [ABSTRACT FROM AUTHOR]