Journal of Microbiology

Review

cAMP Activation of the cAMP Receptor Protein, a Model Bacterial Transcription Factor: Hwan Youn , Marcus Carranza; J. Microbiol. 2023;61(3):277-287. Published online March 9, 2023; DOI: https://doi.org/10.1007/s12275-023-00028-6

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Abstract: The active and inactive structures of the Escherichia coli cAMP receptor protein (CRP), a model bacterial transcr!ption factor, are compared to generate a paradigm in the cAMP-induced activation of CRP. The resulting paradigm is shown to be consistent with numerous biochemical studies of CRP and CRP*, a group of CRP mutants displaying cAMP-free activity. The cAMP affinity of CRP is dictated by two factors: (i) the effectiveness of the cAMP pocket and (ii) the protein equilibrium of apo-CRP. How these two factors interplay in determining the cAMP affinity and cAMP specificity of CRP and CRP* mutants are discussed. Both the current understanding and knowledge gaps of CRP-DNA interactions are also described. This review ends with a list of several important CRP issues that need to be addressed in the future.

Journal Articles

Eradication of drug-resistant Acinetobacter baumannii by cell-penetrating peptide fused endolysin: Jeonghyun Lim , Jaeyeon Jang , Heejoon Myung , Miryoung Song; J. Microbiol. 2022;60(8):859-866. Published online May 25, 2022; DOI: https://doi.org/10.1007/s12275-022-2107-y

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Abstract: Antimicrobial agents targeting peptidoglycan have shown successful results in eliminating bacteria with high selective toxicity. Bacteriophage encoded endolysin as an alternative antibiotics is a peptidoglycan degrading enzyme with a low rate of resistance. Here, the engineered endolysin was developed to defeat multiple drug-resistant (MDR) Acinetobacter baumannii. First, putative endolysin PA90 was predicted by genome analysis of isolated Pseudomonas phage PBPA. The His-tagged PA90 was purified from BL21(DE3) pLysS and tested for the enzymatic activity using Gram-negative pathogens known for having a high antibiotic resistance rate including A. baumannii. Since the measured activity of PA90 was low, probably due to the outer membrane, cell-penetrating peptide (CPP) DS4.3 was introduced at the N-terminus of PA90 to aid access to its substrate. This engineered endolysin, DS-PA90, completely killed A. baumannii at 0.25 μM, at which concentration PA90 could only eliminate less than one log in CFU/ml. Additionally, DS-PA90 has tolerance to NaCl, where the ~50% of activity could be maintained in the presence of 150 mM NaCl, and stable activity was also observed with changes in pH or temperature. Even MDR A. baumannii strains were highly susceptible to DS-PA90 treatment: five out of nine strains were entirely killed and four strains were reduced by 3–4 log in CFU/ml. Consequently, DS-PA90 could protect waxworm from A. baumannii-induced death by ~70% for ATCC 17978 or ~44% for MDR strain 1656-2 infection. Collectively, our data suggest that CPP-fused endolysin can be an effective antibacterial agent against Gramnegative pathogens regardless of antibiotics resistance mechanisms.

Transposon insertion site sequencing (TIS) of Pseudomonas aeruginosa: Hongbaek Cho; J. Microbiol. 2021;59(12):1067-1074. Published online December 4, 2021; DOI: https://doi.org/10.1007/s12275-021-1565-y

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Abstract: Transposon insertion site sequencing (TIS) is a technique that determines the insertion profile of a transposon mutant library by massive parallel sequencing of transposon-genomic DNA junctions. Because the transposon insertion profile reflects the abundance of each mutant in the library, it provides information to assess the fitness contribution of each genetic locus of a bacterial genome in a specific growth condition or strain background. Although introduced only about a dozen years ago, TIS has become an important tool in bacterial genetics that provides clues to study biological functions and regulatory mechanisms. Here, I describe a protocol for generating high density transposon insertion mutant libraries and preparing Illumina sequencing samples for mapping the transposon junctions of the transposon mutant libraries using Pseudomonas aeruginosa as an example.

Development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) thermal inactivation method with preservation of diagnostic sensitivity: Young-Il Kim , Mark Anthony B. Casel , Se-Mi Kim , Seong-Gyu Kim , Su-Jin Park , Eun-Ha Kim , Hye Won Jeong , Haryoung Poo , Young Ki Choi; J. Microbiol. 2020;58(10):886-891. Published online September 29, 2020; DOI: https://doi.org/10.1007/s12275-020-0335-6

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21 Citations

Abstract: Various treatments and agents had been reported to inactivate RNA viruses. Of these, thermal inactivation is generally considered an effective and cheap method of sample preparation for downstream assays. The purpose of this study is to establish a safe inactivation method for SARS-CoV-2 without compromising the amount of amplifiable viral genome necessary for clinical diagnoses. In this study, we demonstrate the infectivity and genomic stability of SARSCoV- 2 by thermal inactivation at both 56°C and 65°C. The
results
substantiate that viable SARS-CoV-2 is readily inactivated when incubated at 56°C for 30 min or at 65°C for 10 min. qRT-PCR of specimens heat-inactivated at 56°C for 30 min or 65°C for 15 min revealed similar genomic RNA stability compared with non-heat inactivated specimens. Further, we demonstrate that 30 min of thermal inactivation at 56°C could inactivate viable viruses from clinical COVID-19 specimens without attenuating the qRT-PCR diagnostic sensitivity. Heat treatment of clinical specimens from COVID-19 patients at 56°C for 30 min or 65°C for 15 min could be a useful
method
for the inactivation of a highly contagious agent, SARS-CoV-2. Use of this method would reduce the potential for secondary infections in BSL2 conditions during diagnostic procedures. Importantly, infectious virus can be inactivated in clinical specimens without compromising the sensitivity of the diagnostic RT-PCR assay.

Georgenia faecalis sp. nov. isolated from the faeces of Tibetan antelope: Xiaoxia Wang , Jing Yang , Yuyuan Huang , Xiaomin Wu , Licheng Wang , Limei Han , Sha Li , Huan Li , Xiaoying Fu , Hai Chen , Xiong Zhu; J. Microbiol. 2020;58(9):734-740. Published online July 24, 2020; DOI: https://doi.org/10.1007/s12275-020-0060-1

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Abstract: Two aerobic, Gram-stain-positive, non-motile, non-sporulating coccoid strains, designated ZLJ0423T and ZLJ0321, were isolated from the faeces of Tibetan antelope (Pantholops hodgsonii). Their optimal temperature, NaCl concentration and pH for growth were 28°C, 0.5% (w/v) NaCl and pH 7.5, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains ZLJ0423T and ZLJ0321 were very similar to each other (99.8%) and had a sequence similarity of 97.0% with Georgenia satyanarayanai NBRC 107612T and Georgenia subflava CGMCC 1.12782T. Phylogenomic analysis based on 688 core genes indicated that these strains formed a clade with G. satyanarayanai NBRC 107612T and Georgenia wutianyii Z294T. The predominant cellular fatty acids were anteiso-C15:0, anteiso-C15:1 A and C16:0. The major menaquinone was MK-8(H4). The cell-wall amino acids consisted of alanine, lysine, glycine and aspartic acid, with lysine as the diagnostic diamino acid. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and two unidentified lipids formed the polar lipid profile. The DNA G + C content of both isolates was 73.9 mol%. The digital DNA–DNA hybridization value between strains ZLJ0423T and ZLJ0321 was 91.2%, but their values with closely related species and other available type strains of the genus Georgenia were lower than the 70% threshold. On the basis of polyphasic taxonomic data, strains ZLJ0423T and ZLJ0321 represent a novel species within the genus Georgenia, for which the name Georgenia faecalis sp. nov. is proposed. The type strain is ZLJ0423T (= CGMCC 1.13681T = JCM 33470T).

Carbohydrate-binding specificities of potential probiotic Lactobacillus strains in porcine jejunal (IPEC-J2) cells and porcine mucin: Valerie Diane Valeriano , Bernadette B. Bagon , Marilen P. Balolong , Dae-Kyung Kang; J. Microbiol. 2016;54(7):510-519. Published online June 28, 2016; DOI: https://doi.org/10.1007/s12275-016-6168-7

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25 Citations

Abstract: Bacterial lectins are carbohydrate-binding adhesins that recognize glycoreceptors in the gut mucus and epithelium of hosts. In this study, the contribution of lectin-like activities to adhesion of Lactobacillus mucosae LM1 and Lactobacillus johnsonii PF01, which were isolated from swine intestine, were compared to those of the commercial probiotic Lactobacillus rhamnosus GG. Both LM1 and PF01 strains have been reported to have good adhesion ability to crude intestinal mucus of pigs. To confirm this, we quantified their adhesion to porcine gastric mucin and intestinal porcine enterocytes isolated from the jejunum of piglets (IPEC-J2). In addition, we examined their carbohydrate-binding specificities by suspending bacterial cells in carbohydrate solutions prior to adhesion assays. We found that the selected carbohydrates affected the adherences of LM1 to IPEC-J2 cells and of LGG to mucin. In addition, compared to adhesion to IPEC-J2 cells, adhesion to mucin by both LM1 and LGG was characterized by enhanced specific recognition of glycoreceptor components such as galactose, mannose, and N-acetylglucosamine. Hydrophobic interactions might make a greater contribution to adhesion of PF01. A similar adhesin profile between a probiotic and a pathogen, suggest a correlation between shared pathogen–probiotic glycoreceptor recognition and the ability to exclude enteropathogens such as Escherichia coli K88 and Salmonella Typhimurium KCCM 40253. These findings extend our understanding of the mechanisms of the intestinal adhesion and pathogen-inhibition abilities of probiotic Lactobacillus strains.

Research Support, Non-U.S. Gov'ts

Development of a stringent ELISA protocol to evaluate anti-viral hemorrhagic septicemia virus-specific antibodies in olive flounder Paralichthys olivaceus with improved specificity: Hyoung Jun Kim , Jeong Su Park , Se Ryun Kwon; J. Microbiol. 2015;53(7):481-485. Published online June 27, 2015; DOI: https://doi.org/10.1007/s12275-015-5101-9

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9 Citations

Abstract: Olive flounder were vaccinated with polyinosinic:polycytidylic acid [Poly (I:C)] to prevent viral hemorrhagic septicemia (VHS). Vaccine efficacy was verified by detection of anti- VHS virus (VHSV) antibodies using enzyme-linked immunosorbent assay (ELISA). In the study, ELISA absorbance values of the negative control group [Poly (I:C)-MEM10] were saturated when an ELISA protocol, that includes pretreatment of the fish sera with 5% skim milk, was used. However, the saturated OD values in the negative control did not correlate with a specific immune response against VHSV, because the group showed low survival rate (only 10%) following the VHSV challenge. Also, OD values of Poly (I:C)- VHSV group were high, and the group showed high survival rate (97.5%) against VHSV challenge test. It was suggested that the high OD values were possibly due to the presence of anti-fetal bovine serum (FBS) cross-reactivity. To compensate this, we subtracted the absorbance of infectious hematopoietic necrosis (IHNV)-Ag plates from those of the VHSV-Ag plates. However, the average value for the Poly (I:C)-VHSV group (0.167) was lower than expected even though high survival rate. We used an advanced ELISA system to pre-treat fish sera with 5% skim milk and two novirhabdoviruses as capture antigens as well as 50% FBS. The corrected absorbance values for pre-treated fish sera from the negative control Poly (I:C)-MEM10 and experimental Poly (I:C)-VHSV groups averaged 0.033 and 0.579, respectively. The specific VHSV antibody response of the vaccinated group was assessed using fish sera pretreated with skim milk and FBS and by calculating the corrected absorbance values from ELISA with two novirhabdovirus capture antigens.

Effect of promoter-upstream sequence on σ38-dependent stationary phase gene transcription: Hyung-Ju Lim , Kwangsoo Kim , Minsang Shin , Jae-Ho Jeong , Phil Youl Ryu , Hyon E. Choy; J. Microbiol. 2015;53(4):250-255. Published online April 8, 2015; DOI: https://doi.org/10.1007/s12275-015-4681-8

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Abstract: σ38 in Escherichia coli is required for expression of a subset of stationary phase genes. However, the promoter elements for σ38-dependent genes are virtually indistinguishable from that for σ70-dependent house-keeping genes. hdeABp is a σ38-dependent promoter and LEE5p is a σ70-dependent promoter, but both are repressed by H-NS, a bacterial histone- like protein, which acts at promoter upstream sequence. We swapped the promoter upstream sequences of the two promoters and found that the σ dependency was switched. This was further verified using lacUV5 core promoter. The
results
suggested that the determinant for σ38-dependent promoter lies in the promoter upstream sequence.

Trans-Membrane Transport of n-Octadecane by Pseudomonas sp. DG17: Fei Hua , Hong Qi Wang , Yi Li , Yi Cun Zhao; J. Microbiol. 2013;51(6):791-799. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3259-6

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23 Citations

Abstract: The trans-membrane transport of hydrocarbons is an important and complex aspect of the process of biodegradation of hydrocarbons by microorganisms. The mechanism of transport of 14C n-octadecane by Pseudomonas sp. DG17, an alkane-degrading bacterium, was studied by the addition of ATP inhibitors and different substrate concentrations. When the concentration of n-octadecane was higher than 4.54 μmol/L, the transport of 14C n-octadecane was driven by a facilitated passive mechanism following the intra/extra substrate concentration gradient. However, when the cells were grown with a low concentration of the substrate, the cellular accumulation of n-octadecane, an energy-dependent process, was dramatically decreased by the presence of ATP inhibitors, and n-octadecane accumulation continually increased against its concentration gradient. Furthermore, the presence of non-labeled alkanes blocked 14C n-octadecane transport only in the induced cells, and the trans-membrane transport of n-octadecane was specific with an apparent dissociation constant Kt of 11.27 μmol/L and Vmax of 0.96 μmol/min/mg protein. The results indicated that the transmembrane transport of n-octadecane by Pseudomonas sp. DG17 was related to the substrate concentration and ATP.

NOTE] The Activity of Phosphoinositide-Specific Phospholipase C Is Required for Vegetative Growth and Cell Wall Regeneration in Coprinopsis cinerea: Young Taek Oh , Chun-Seob Ahn , Kyung-Jin Lee , Jeong-Geun Kim , Hyeon-Su Ro , Jae Won Kim , Chang-Won Lee; J. Microbiol. 2012;50(4):689-692. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2004-x

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Abstract: Three isotypes of phosphoinositide-specific phospholipase C designated CcPLC1, CcPLC2, and CcPLC3 were identified in Coprinopsis cinerea, through a search of the genome sequence database. The functional role of the PI-PLCs were studied by using U73122, which specifically inhibits the activity of PI-PLC. The specificity of the inhibitor effect was confirmed by using an inactive structural analog U73433. The inhibition of PI-PLCs activity resulted in severely retarded germination of basidiospores and oidia, reduced hyphal growth, knobbly hyphal tips with many irregular side branches, and aberrant (branch-like structure) clamp cells. Furthermore, U73122 definitely inhibited cell wall formation. Here we report that PI-PLCs play important roles in various aspects of C. cinerea biology.

A Genome-Wide Identification of Genes Potentially Associated with Host Specificity of Brucella Species: Kyung Mo Kim , Kyu-Won Kim , Samsun Sung , Heebal Kim; J. Microbiol. 2011;49(5):768-775. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1084-3

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Abstract: Brucella species are facultative intracellular pathogenic α-Proteobacteria that can cause brucellosis in humans and domestic animals. The clinical and veterinary importance of the bacteria has led to well established studies on the molecular mechanisms of Brucella infection of host organisms. However, to date, no genome-wide study has scanned for genes related to the host specificity of Brucella spp. The majority of bacterial genes related to specific environmental adaptations such as host specificity are well-known to have evolved under positive selection pressure. We thus detected signals of positive selection for individual orthologous genes among Brucella genomes and identified genes related to host specificity. We first determined orthologous sets from seven completely sequenced Brucella genomes using the Reciprocal Best Hits (RBH). A maximum likelihood analysis based on the branch-site test was accomplished to examine the presence of positive selection signals, which was subsequently confirmed by phylogenetic analysis. Consequently, 12 out of 2,033 orthologous genes were positively selected by specific Brucella lineages, each of which belongs to a particular animal host. Extensive literature reviews revealed that half of these computationally identified genes are indeed involved in Brucella host specificity. We expect that this genome-wide approach based on positive selection may be reliably used to screen for genes related to environmental adaptation of a particular species and that it will provide a set of appropriate candidate genes.

NOTE] Development of Porphyromonas gingivalis-Specific Quantitative Real-Time PCR Primers Based on the Nucleotide Sequence of rpoB: Soon-Nang Park , Jae-Yoon Park , Joong-Ki Kook; J. Microbiol. 2011;49(2):315-319. Published online May 3, 2011; DOI: https://doi.org/10.1007/s12275-011-1028-y

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17 Citations

Abstract: Species-specific quantitative real-time PCR (qPCR) primers were developed for the detection of Porphyromonas gingivalis. These primers, Pg-F/Pg-R, were designed based on the nucleotide sequences of RNA polymerase β-subunit gene (rpoB). Species-specific amplicons were obtained from the tested P. gingivalis strains but not in any of the other strains (46 strains of 46 species). The qPCR primers could detect as little as 4 fg of P. gingivalis chromosomal DNA. These findings suggest that these qPCR primers are suitable for applications in epidemiological studies.

Distinctive Endophytic Fungal Assemblage in Stems of Wild Rice (Oryza granulata) in China with Special Reference to Two Species of Muscodor (Xylariaceae): Zhi-lin Yuan , Zhen-zhu Su , Li-juan Mao , Yang-qing Peng , Guan-mei Yang , Fu-cheng Lin , Chu-long Zhang; J. Microbiol. 2011;49(1):15-23. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0213-3

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30 Citations

Abstract: Ecological niches in the rhizosphere and phyllosphere of grasses capable of sustaining endophytes have been extensively studied. In contrast, little information regarding the identity and functions of endophytic fungi in stems is available. In this study, we investigated the taxonomic affinities, diversity, and host specificities of culturable endophytes in stems of wild rice (Oryza granulata) in China. Seventy-four isolates were recovered. Low recovery rate (11.7%) indicated that there were relatively few sites for fungal infection. Identification using morphology, morphospecies sorting, and molecular techniques resulted in classification into 50 taxa, 36 of which were recovered only once. Nucleotide sequence similarity analysis indicated that 30% of the total taxa recovered were highly divergent from known species and thus may represent lineages new to science. Most of the taxa were classified as members of the classes Sordariomycetes or Dothideomycetes (mainly in Pleosporales). The presence of Arthrinium and Magnaporthaceae species, most often associated with poaceous plants, suggested a degree of host specificity. A polyphasic approach was employed to identify two Muscodor taxa based on (i) ITS and RPB2 phylogenies, (ii) volatile compounds produced, and (iii) an in vitro bioassay of antifungal activity. This to our knowledge is only the second report regarding the isolation of Muscodor spp. in China. Therefore, we hypothesize that wild plants represent a huge reservoir of unknown fungi. The prevalence, novelty, and species-specificity of unique isolates necessitate a reevaluation of their contribution to ecosystem function and fungal biodiversity.

Evaluation of the Sensitivity and Specificity of Primer Pairs and the Efficiency of RNA Extraction Procedures to Improve Noroviral Detection from Oysters by Nested Reverse Transcription-Polymerase Chain Reaction: Cheonghoon Lee , Sooryun Cheong , Hee-Jung Lee , Miye Kwon , Ilnam Kang , Eun-Gyoung Oh , Hong-Sik Yu , Soon-Bum Shin , Sang-Jong Kim; J. Microbiol. 2010;48(5):586-593. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0047-4

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6 Citations

Abstract: Noroviruses (NoV) are the key cause of acute epidemic gastroenteritis, and oysters harvested from NoVpolluted sea areas are considered as the significant vectors of viral transmission. To improve NoV detection from oyster using nested reverse transcription-polymerase chain reaction (RT-PCR), we evaluated the sensitivity and specificity of previously published primer pairs and the efficiency of different RNA extraction procedures. Among the primer pairs used for RT-PCR, the sensitivity of GIF1/GIR1-GIF2/GIR1 and GIIF1/GIIR1-GIIF2/GIIR1 was higher than that of other primer pairs used in nested RT-PCR for the detection of NoV genogroup I (NoV GI) and NoV GII from both NoV-positive stool suspension and NoVseeded oyster concentrates, respectively; the resulting products showed neither unspecific bands in the positive samples nor false-positive bands in the negative controls. The extraction of NoV RNA from oyster samples using a QIAamp? Viral RNA Mini kit with a QIAshredderTM Homogenizer pretreatment afforded more efficient recovery (mean recovery for NoV GI and GII, 6.4%) and the procedure was less time consuming (<30 min) than most other RNA extraction procedures. The results of RNA extraction procedure and primer pairs evaluated by nested RT-PCR assay in this study can be useful for monitoring NoV contamination in oysters, which is an indicator of possible public health risks.

Development of a Latex Agglutination Test for Norovirus Detection: Heetae Lee , YoungBin Park , Misoon Kim , Youngmee Jee , Doo-sung Cheon , Hae Sook Jeong , GwangPyo Ko; J. Microbiol. 2010;48(4):419-425. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0071-4

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Abstract: Norovirus (NoV) is the leading cause of acute gastroenteritis worldwide. Currently, reverse transcription polymerase chain reaction (RT-PCR) is used commonly to detect NoVs in both clinical and environmental samples. However, RT-PCR requires expensive equipment and cannot be performed on site. In this study, a latex agglutination test (LAT) using antibody-labeled latex beads for detecting NoVs was developed. Two kinds of polyclonal antibodies, one generated from synthetic peptides and the other from E. coli-expressed NoV capsid proteins, were used to develop the LAT. Each of these polyclonal antibodies was immobilized on the surface of latex beads and tested for the ability to detect NoVs. Under optimized conditions, our LAT detected GII.4 NoV at concentrations as low as 3.3×105 RT-PCR units/ml in stool samples. The detection limit for the LAT was approximately 1.7×103 RT-PCR units. Forty-eight stool samples were tested for NoVs using this LAT. In comparison with an RT-PCR assay, the sensitivity and specificity of the LAT were 35% and 100%, respectively. With further optimization, this LAT used with appropriate antibodies could be applied for convenient detection of NoVs in clinical diagnosis and food monitoring.

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