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Type VI secretion system (T6SS) is a contact-dependent secretion system, employed by most gram-negative bacteria for translocating effector proteins to target cells. The present study was conducted to investigate T6SS in Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight in rice, and to unveil its functions. Two T6SS clusters were found in the genome of Xoo PXO99A. The deletion mutants, Δhcp1, Δhcp2, and Δhcp12, targeting the hcp gene in each cluster, and a double-deletion mutant targeting both genes were constructed and tested for growth rate, pathogenicity to rice, and inter-bacterial competition ability. The results indicated that hcp in T6SS-2, but not T6SS-1, was involved in bacterial virulence to rice plants. However, neither T6SS-1 nor T6SS-2 had any effect on the ability to compete with Escherichia coli or other bacterial cells. In conclusion, T6SS gene clusters in Xoo have been characterized, and its role in virulence to rice was confirmed.

In this study, two bacterial strains, IRP7 and IRP8, were selected to induce resistance against pine wilt disease (PWD). Foliar application with these strains to nematode-inoculated pine seedlings significantly reduced PWD severity. The effect of nematode inoculation and bacterial treatment on the rhizosphere bacterial community was investigated. The results indicated that the rhizosphere of nematode-inoculated seedlings contained a lower relative abundance of beneficial microbes such as Paraburkholderia, Bradyrhizobium, Rhizobacter, Lysobacter, and Caballeronia. Bacterial treatment resulted in significant changes in the microbes that were represented in relatively low relative abundance. Treatment with IRP7 resulted in an increase in the relative abundance of Nitrospirillum, Bacillus, and Luteibacter, which might be useful for protection against infection. Treatment with IRP8 resulted in an increase in the relative abundance of obligate bacterial predators of the Bdellovibrio genus that were previously shown to control several bacterial phytopathogens and may have a role in the management of nematode-carried bacteria. The selected bacteria were identified as Pseudomonas koreensis IRP7 and Lysobacter enzymogenes IRP8 and are suggested as a potential treatment for induced resistance against PWD. To our knowledge, this is the first report on the effect of foliar treatment with resistance-inducing bacteria on the rhizosphere microbiota.

The partner switching system (PSS) of the SigF regulatory pathway in Mycobacterium smegmatis has been previously demonstrated to include the anti-sigma factor RsbW (MSMEG_1803) and two anti-sigma factor antagonists RsfA and RsfB. In this study, we further characterized two additional RsbW homologs and revealed the distinct roles of three RsbW homologs [RsbW1 (MSMEG_1803), RsbW2 (MSMEG_6129), and RsbW3 (MSMEG_1787)] in the SigF PSS. RsbW1 and RsbW2 serve as the anti-sigma factor of SigF and the protein kinase phosphorylating RsfB, respectively, while RsbW3 functions as an anti-SigF antagonist through its protein interaction with RsbW1. Using relevant mutant strains and RNA sequencing analysis, RsfB was demonstrated to be the major anti-SigF antagonist in M. smegmatis. The phosphorylation state of Ser-63 was shown to determine the functionality of RsfB as an anti-SigF antagonist. RsbW2 was demonstrated to be the only protein kinase that phosphorylates RsfB in M. smegmatis. Phosphorylation of Ser-63 inactivates RsfB to render it unable to interact with RsbW1. Our comparative RNA sequencing analysis of the wild-type strain of M. smegmatis and its isogenic aa3 mutant strain lacking the aa3 cytochrome c oxidase of the respiratory electron transport chain revealed that expression of the SigF regulon is strongly induced under respiration-inhibitory conditions in an RsfB-dependent way.

The pinewood nematode (PWN) Bursaphelenchus xylophilus causes pine wilt disease, which results in substantial economic and environmental losses across pine forests worldwide. Although systemic acquired resistance (SAR) is effective in controlling PWN, the detailed mechanisms underlying the resistance to PWN are unclear. Here, we treated pine samples with two SAR elicitors, acibenzolar-S-methyl (ASM) and methyl salicylic acid (MeSA) and constructed an in vivo transcriptome of PWN-infected pines under SAR conditions. A total of 252 million clean reads were obtained and mapped onto the reference genome. Compared with untreated pines, 1091 and 1139 genes were differentially upregulated following the ASM and MeSA treatments, respectively. Among these, 650 genes showed co-expression patterns in response to both SAR elicitors. Analysis of these patterns indicated a functional linkage among photorespiration, peroxisome, and glycine metabolism, which may play a protective role against PWN infection-induced oxidative stress. Further, the biosynthesis of flavonoids, known to directly control parasitic nematodes, was commonly upregulated under SAR conditions. The ASM- and MeSA-specific expression patterns revealed functional branches for myricetin and quercetin production in flavonol biosynthesis. This study will enhance the understanding of the dynamic interactions between pine hosts and PWN under SAR conditions.

Pine wilt disease (PWD) caused by Bursaphelenchus xylophilus is a major threat to pine forests worldwide. Induction of resistance is a promising and safe management option that should be investigated in relation to its possible influence on the pine tree ecosystem, including the surrounding microbial communities. In this study, two main resistance-inducing chemical elicitors, methyl salicylic acid (MeSA) and acibenzolar-s-methyl (ASM), were tested for their control efficiency against PWD and their influence on the rhizosphere microbial composition. Foliar treatment of pine seedlings with the chemical elicitors resulted in a reduction in PWD severity, with ASM showing better control efficacy, reaching up to 73% compared to the untreated control. Moreover, bacterial community analysis of the rhizosphere revealed significant changes in several microbial taxa that were present at low relative abundance. In particular, ASM treatment resulted in a significant increase in specific microbial taxa, including members of the Rhodanobacter, Devosia, Bradyrhizobium, Acidibacter, Mesorhizobium, and Hyphomicrobium genera, which are known to play ecological and plant growth-promoting roles. Furthermore, chitinolytic bacteria were shown to be reduced in response to treatment with both MeSA and ASM. Altogether, the present findings demonstrate the occurrence of significant alterations in several ecologically important microbial taxa after treatment with resistance-inducing chemicals. As compared to MeSA treatment, ASM treatment was more effective at suppressing PWD and resulted in more beneficial changes in rhizosphere microbial composition.

CK2α is a constitutively active and highly conserved serine/threonine protein kinase that is involved in the regulation of key cellular metabolic pathways and associated with a variety of tumours and cancers. The most well-known CK2α inhibitor is the human clinical trial candidate CX-4945, which has recently shown to exhibit not only anti-cancer, but also anti-fungal properties. This prompted us to work on the CK2α orthologue, Cka1, from the pathogenic fungus Cryptococcus neoformans, which causes life-threatening systemic cryptococcosis and meningoencephalitis mainly in immunocompromised individuals. At present, treatment of cryptococcosis remains a challenge due to limited anti-cryptococcal therapeutic strategies. Hence, expanding therapeutic options for the treatment of the disease is highly clinically relevant. Herein, we report the structures of Cka1-AMPPNP-Mg2+ (2.40 Å) and Cka1-CX-4945 (2.09 Å). Structural comparisons of Cka1-AMPPNP-Mg2+ with other orthologues revealed the dynamic architecture of the N-lobe across species. This may explain for the difference in binding affinities and deviations in protein-inhibitor interactions between Cka1-CX-4945 and human CK2α-CX-4945. Supporting it, in vitro kinase assay demonstrated that CX-4945 inhibited human CK2α much more efficiently than Cka1. Our results provide structural insights into the design of more selective inhibitors against Cka1.

SummaryTelomeric repeats located within chromosomes are called interstitial telomeric sequences (ITSs). They are polymorphic in length and are likely hotspots for initiation of chromosomal rearrangements that have been linked to human disease. Using our S. cerevisiae system to study repeat-mediated genome instability, we have previously shown that yeast telomeric (Ytel) repeats induce various gross chromosomal rearrangements (GCR) when their G-rich strands serve as the lagging strand template for replication (G orientation). Here, we show that interstitial Ytel repeats in the opposite C orientation prefer to expand rather than cause GCR. A tract of eight Ytel repeats expands at a rate of 4 × 10−4 per replication, ranking them among the most expansion-prone DNA microsatellites. A candidate-based genetic analysis implicates both post-replication repair and homologous recombination pathways in the expansion process. We propose a model for Ytel repeat expansions and discuss its applications for genome instability and alternative telomere lengthening (ALT).

The nuclear protein HMGB1 (high mobility group box 1) is secreted by monocytes-macrophages in response to inflammatory stimuli and serves as a danger-associated molecular pattern. Acetylation and phosphorylation of HMGB1 are implicated in the regulation of its nucleocytoplasmic translocation for secretion, although inflammatory stimuli are known to induce H2O2 production. Here we show that H2O2-induced oxidation of HMGB1, which results in the formation of an intramolecular disulfide bond between Cys23 and Cys45, is necessary and sufficient for its nucleocytoplasmic translocation and secretion. The oxidation is catalyzed by peroxiredoxin I (PrxI) and PrxII, which are first oxidized by H2O2 and then transfer their disulfide oxidation state to HMGB1. The disulfide form of HMGB1 showed higher affinity for nuclear exportin CRM1 compared with the reduced form. Lipopolysaccharide (LPS)–induced HMGB1 secretion was greatly attenuated in macrophages derived from PrxI or PrxII knockout mice, as was the LPS-induced increase in serum HMGB1 levels. Keywords: HMGB1, Oxidation, Secretion, Peroxiredoxin, H2O2

Azolla Lam. is a small aquatic fern with deeply bilobed leaves, which are consisted of a thick greenish, withchlorophyll, upper (dorsal) lobe and a thinner, translucent lower (ventral) lobe, without chlorophyll,submerged in the water. Azolla blight was observed at a lotus pond. Mycological characteristics of the fungusassociated with Azolla blight was immediately determined as Rhizoctonia sp. by the thickness and branchingof hypha at right angles at the point toward the distal end of septa, with branching hypha is constricted. Thefungus produced brown mycelia and dark brown sclerotia on PDA. The optimum temperature for mycelialgrowth and sclerotia formation were 25oC and 30oC, respectively. The optimum temperature for fungalinfection was 30oC, when spray inoculated. Phylogenetic analysis of rDNA-ITS revealed that the fungus wasidentified as Rhizoctonia solani (AG-1 IA) closest to one causing rice sheath blight disease. This is the firstreport on the blight disease of Azolla caused by R. solani in Korea.

Aims: To evaluate whether the gut bacteria of insecticide-resistant, insecticide-susceptible and field-caught populations of the lepidopteran insect pest diamondback moth (DBM) –Plutella xylostella (L.) – are variable and their role in host protection and nutrition. Methods and Results: The gut bacterial populations of the three DBM larvae populations were found to be significantly different, irrespective of the developmental stage. The 16S rRNA gene sequence analysis of the DBM gut bacteria revealed that the bacterial population from the prothiofos-resistant larval gut was more diversified with Pseudomonas sp., Stenotrophomonas sp., Acinetobacter sp., and Serratia marcescens. Meanwhile, the susceptible larvae were associated with Brachybacterium sp., Acinetobacter sp. and S. marcescens and the field-caught population harboured a rather simple gut microflora of phylotypes belonging to Serratia. The siderophore-producing Pseudomonas sp. strain PRGB06 showed antagonistic activity towards entomopathogenic fungi, including Beaveria bassiana, Hirsutella thompsonii, Metarhizium anisopliae, Paecilomyces sp., and Paecilomyces tenuipes, while the chitinase-producing S. marcescens enhanced the larval growth and development. Conclusion: There was a significant variation in the gut bacteria from the three different populations of DBM. The production of antifungal siderophore compounds, like pyoverdine, may contribute to host antagonism against entomopathogens. The production of chitinase by gut bacteria appeared to contribute to host nutrition. Significance and Impact of the Study: The results provide the first comprehensive description of the gut microbial communities in three different populations of an important crucifer pest DBM and suggest that the bacteria associated with the insect pest could be of interest for developing a pest management strategy.

Mating behavior of the pine sawyer, Monochamus saltuarius Gebier (Cerambycidae: Lamiinae) was observed on pine trees in an outdoor net cage kept in one healthy pine tree and a cluster of pinelogs (cut over one month) to attract the beetles. When sawyer adult pairs released into net cage, they all moved to the pinelogs. And then the male was staying motionless with his antenna outstretched while the female was actively moving in his vicinity. The mating behavioral reactions were followed by the next steps: The first phase of courtship was initiated by the female approach toward the male. Second, male dashed, contacted antenna and mounted female's back. Third, female carried the male on her back and walked around and then male began to lick. Fourth, male inserted his penis into the female's genitalia when female stopped walking. While a long pairbond, the mating pair repeated copulations, lastly, when mating is over, male and female stayed separately. The mating began on sunset and resting of both male and female began at the temperature below about 20 °C. Substantially straightforward mate searching by female indicates the presence of a male sex pheromone.

Laboratory experiment was conducted to compare the relative toxicity of biopesticides like Pseudomonas fluorescens strain pfl and neem oil with imidacloprid, quinalphos, and endosulfan against an egg parasitoid, Trichogramma chilonis, and a predatory green lacewing, Chrysoperla carnea. Biopesticides were safer than chemical insecticides in minimizing harmful effects on development and behavior of two natural enemies. P. fluorescens treatment recorded high parasitism (≈ 73 %) and parasitoid egg development (≈ 72 %) of T. chilonis. It also gave high egg (≈ 75 %) development on C. carnea. Neem oil recorded 58.9 % parasitoid eme-crgence, and 59.3 % parasitism, and 63.1 % egg hat-hability. Other chemical insecticides exhibited high damages on the natural enemies.

Interspecies variations and mutations associated with rifampin resistance in rpoB of Mycobacterium allow for the simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by PCR-SSCP analysis and PCR- sequencing. One hundred and ten strains of rifampin-susceptible M. tuberculosis, 14 strains of rifampin-resistant M. tuberculosis, and four strains of the M. avium complex were easily identified by PCR-SSCP. Of another seven strains, which showed unique SSCP patterns, three were identified as rifampin-resistant M. tuberculosis and four as M. terrae complex by subsequent sequence analysis of their rpoB DNAs (306 bp). These results were concordant with those obtained by susceptibility testing, biochemical identification, and 16S rDNA sequencing.

A novel duplex PCR method that can amplify the 235- and 136-bp rpoB DNAs of Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM), respectively, with two different sets of primers was used to differentially identify 44 reference strains and 379 clinical isolates of mycobacteria in a single-step assay. Showing 100% sensitivity and specificity, the duplex PCR method could clearly differentiate M . tuberculosis complex and NTM strains. In addition, restriction fragment length polymorphism analysis and direct sequencing of the amplicon of NTM could be used to supplement species identification.

Either PCR-mediated single strand conformation polymorphism (SSCP) analysis or DNA sequencing of rpoB DNA (157 bp) can be used as a rapid screening method for the detection of mutations related to the rifampin resistance of Mycobacterium tuberculosis . However, due to the nonspecific amplification of rpoB DNA from nontuberculous mycobacteria these methods cannot be directly applied to clinical specimens such as sputa. We developed a nested PCR method that can specifically amplify the rpoB DNA of M. tuberculosis on the basis of rpoB DNA sequences of 44 mycobacteria. Nested PCR-linked SSCP analysis and the DNA sequencing method were applied directly in order to detect M. tuberculosis and determine its rifampin susceptibility in 56 sputa. The results obtained by nested PCR-SSCP and DNA sequencing were concordant with those of conventional drug susceptibility testing and DNA sequencing performed with culture isolates.

A previously undescribed, slowly growing, scotochromogenic mycobacterium was isolated from a patient with symptomatic pulmonary infection during hsp65 sequence-based identification of Korean clinical isolates. Phenetic characteristics of this strain were generally similar to those of Mycobacterium nebraskense and Mycobacterium scrofulaceum. However, some phenetic characteristics differentiated it from these two species. Its 16S rRNA gene sequences were unique and phylogenetic analysis based on 16S rRNA gene sequences placed the organism in the slowly growing Mycobacterium group close to M. nebraskense and M. scrofulaceum. Its unique mycolic acid profiles and the results of phylogenetic analysis based on two independent alternative chronometer molecules, hsp65 and rpoB, confirmed the taxonomic status of this strain as representing a novel species. These data support the conclusion that this strain represents a novel mycobacterial species, for which the name Mycobacterium seoulense sp. nov. is proposed. The type strain is strain 03-19T (=DSM 44998T=KCTC 19146T).

To evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 gene for the direct detection of pathogenic mycobacteria from clinical specimens, we applied this method to 40 sputum samples and compared the results to those obtained by IS 6110 PCR. Although this method showed a sensitivity slightly lower than IS 6110 PCR (97.5% vs. 100%), it detected infections of M. avium complex (MAC) in two patients, which was not possible by IS 6110 PCR. We conclude that AvaII PRA is a highly effective method for directly detecting pathogenic mycobacteria in primary clinical specimens.

Tuberculosis caused by mycobacteria, mainly Mycobacterium tuberculosis, is a major infectious disease of the respiratory system. An early diagnosis followed by chemotherapy is the major control strategy. In an effort to identify the antigens suitable for immunodiagnosis and vaccines, the proteins secreted in a culture medium from the M. tuberculosis K-strain, which is the most prevalent among the clinical isolates in Korea and belongs to the Beijing family, were analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and compared with those from the M. tuberculosis H37Rv and CDC1551 strains. Eight proteins, Rv0652, Rv1636, Rv2818c, Rv3369, Rv3865, Rv0566c, MT3304, and Rv3160, were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and found to be relatively abundant in the culture medium from the M. tuberculosis K-strain but less so from the CDC1551 or H37Rv strains. In addition, Rv3874 (CFP-10), Rv-0560c and Rv3648c, which were expressed increasingly in the K and CDC1551 strains, were also identified using the same proteomics technology. All proteins were prepared by molecular cloning, expression in Escherichia coli followed by affinity purification. Among them, three proteins, rRv3369, rRv0566c, and rRv3874, were selected by prescreening and examined for their potential as serodiagnostic antigens using an enzyme-linked immunosorbent assay. When 100 sera from tuberculosis patients and 100 sera from the healthy controls were analyzed, rRV3369, rRv3874, and rRv0566c showed a sensitivity of 60%, 74%, and 43%, and a specificity of 96%, 97%, and 84%, respectively. These results suggest that the rRv3369 and rRv3874 proteins, which were expressed more abundantly in the more recently obtained clinical isolates of M. tuberculosis than in the laboratory-adapted H37Rv strain, are promising for use in the serodiagnosis of tuberculosis.

A reverse probe hybridization method using two different Mycobacterium tuberculosis-specific rpoB DNA probes in combination was evaluated for the identification of M. tuberculosis culture isolates. Among the 384 isolates tested, 354 strains were identified as M. tuberculosis, which included 37 rifampin-resistant strains, and 30 were nontuberculous mycobacteria (NTM). This result was in accord with partial rpoB sequence analysis and IS6110 polymerase chain reaction (PCR) results, but not with the results of biochemical testing, which produced two false negative results. Because of its high level of sensitivity and specificity, we suggest that M. tuberculosis-specific rpoB probes immobilized on micro-titer well plates or on other solid matrixes can be used efficiently for the rapid and convenient identification of M. tuberculosis.

Resistance to isoniazid in Mycobacterium tuberculosis has been associated with mutations in genes encoding the mycobacterial catalase-peroxidase (katG) and the InhA protein (inhA). Among the 26 isoniazid-resistant clinical isolates evaluated in this study, mutations in putative inhA regulatory sequences were identified in 2 catalase-positive isolates, katG gene alterations were detected in 20 strains, and 4 isolates had wild-type katG and inhA genes. Mutations in the katG gene were detected in all 11 catalase-negative isolates: one frameshift insertion, two partial gene deletions, and nine different missense mutations were identified. An arginine-to-leucine substitution at position 463 was detected in nine catalase-positive isolates. However, site-directed mutagenesis experiments demonstrated that the presence of a leucine at codon 463 did not alter the activity of the M. tuberculosis catalase-peroxidase and did not affect the capacity of this enzyme to restore isoniazid susceptibility to isoniazid-resistant, KatG-defective Mycobacterium smegmatis BH1 cells. These studies further support the association between katG and inhA gene mutations and isoniazid resistance in M. tuberculosis, while also suggesting that other undefined mechanisms of isoniazid resistance exist.

The molecular mechanisms of resistance to streptomycin, rifampin, and isoniazid in 53 Mycobacterium tuberculosis clinical isolates were examined. Twenty-five of 44 streptomycin-resistant strains had mutations in the rpsL gene and 5 of these had rrs gene perturbations. The region of the rpoB gene that is associated with resistance to rifampin was altered in 28 of 29 rifampin-resistant strains. Mutations in known genetic markers of isoniazid resistance were detected in 25 of 42 isoniazid-resistant isolates: 20 strains had katG gene alterations and 5 had perturbations in the inhA operon. Of the 20 multiply resistant strains with reduced sensitivity to streptomycin, rifampin, and isoniazid, 11 had mutations in genetic markers associated with resistance to each of these three drugs. These studies suggest that the primary mechanism of multiple drug resistance in tuberculosis is the accumulation of mutations in individual drug target genes.

Purpose: With increasing frequency, Yersinia enterocolitica is being recognized as an important bacterial cause of acute gastrointestinal infection with abdominal pain. In addition, the association of Y. enterocolitica infections with acute appendicitis has been suggested. This study was undertaken to ascertain whether Y. enterocolitica is a possible infectious agent in acute appendicitis. Methods: Between December 2007 and April 2008, 162 patients who underwent appendectomy for presumed appendicitis, enrolled in this prospective study. After surgical excision of appendix, a portion of each specimen was cultured for Y. enterocolitica with highly selective media (Cefsulodin-Irgasan-Novobiocin agar). Results: Pathologically, 150 of the patients had appendicitis and 12 patients had normal appendices. Only one of the 150 patients (0.7%) with appendicitis was found to be culture positive for Y. enterocolitica, while it was not detected from normal appendices. Conclusion: The authors were unable to implicate Y. enterocolitica as a major pathogen in acute appendicitis within the Seoul area. However, we thought there to be more need for investigation for association of Y. enterocolitica with acute appendicitis over a broader area and season.

The goals of this study were to identify first-line drug resistance in new and previously treated tuberculosis (TB) cases and to determine risk factors for multidrug resistant TB (MDR-TB) at a private referral center in Korea. All patients with culture confirmed pulmonary TB over a 2-yr period between July 2002 and June 2004 were prospectively included in this study. In total, 637 patients were included; 512 (80.4%) were new cases, and 125 (19.6%) were previously treated cases. Resistance to at least one first-line drug was identified in 11.7% of new cases and 41.6% of previously treated cases. MDR-TB was detected in 3.9% of new cases and 27.2% of previously treated cases. The proportion of extensively drug-resistant TB among MDR-TB patients was 16.7% (9/54). Factors associated with MDR-TB included age under 45 yr, previous TB treatment, and the presence of cavitation on chest radiography. Rates of first-line drug resistance are high, particularly in previously treated patients, in the private sector in Korea. This underscores the need for an improved control program, coupled with early diagnosis of MDR-TB, to reduce the spread and development of resistance.