Journal of Microbiology

Journal Articles

Cytophaga hutchinsonii chu_2177, encoding the O-antigen ligase, is essential for cellulose degradation: Yahong Tan , Wenxia Song , Lijuan Gao , Weican Zhang , Xuemei Lu; J. Microbiol. 2022;60(4):364-374. Published online January 7, 2022; DOI: https://doi.org/10.1007/s12275-022-1531-3

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Abstract: Cytophaga hutchinsonii can efficiently degrade crystalline cellulose, in which the cell surface cellulases secreted by the type IX secretion system (T9SS) play important roles, but the degradation mechanism remains unclear, and the anchor mechanism of cellulases on the outer membrane in C. hutchinsonii has not been studied. Here, chu_2177 was identified by transposon mutagenesis and was proved to be indispensable for cellulose utilization in C. hutchinsonii. Disruption of chu_2177 resulted in O-antigen deficiency and chu_ 177 could confer O-antigen ligase activity upon an Escherichia coli waal mutant, indicating that chu_2177 encoded the Ontigen ligase. Moreover, deletion of chu_2177 caused defects in cellulose utilization, cell motility, biofilm formation, and stress resistance. Further study showed that the endoglucanase activity was markedly decreased in the outer membrane but was increased in the culture fluid without chu_2177. Western blot proved that endoglucanase CHU_1336 was not located on the outer membrane but was released in the culture fluid of the Δ2177 mutant. Further proteomics analysis showed that many cargo proteins of T9SS were missing in the outer membrane of the Δ2177 mutant. Our study revealed that the deletion of chu_2177 affected the localization of many T9SS cargo proteins including cellulases on the outer membrane of C. hutchinsonii.

The effects of deletion of cellobiohydrolase genes on carbon source-dependent growth and enzymatic lignocellulose hydrolysis in Trichoderma reesei: Meibin Ren , Yifan Wang , Guoxin Liu , Bin Zuo , Yuancheng Zhang , Yunhe Wang , Weifeng Liu , Xiangmei Liu , Yaohua Zhong; J. Microbiol. 2020;58(8):687-695. Published online June 10, 2020; DOI: https://doi.org/10.1007/s12275-020-9630-5

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Abstract: The saprophytic fungus Trichoderma reesei has long been used as a model to study microbial degradation of lignocellulosic biomass. The major cellulolytic enzymes of T. reesei are the cellobiohydrolases CBH1 and CBH2, which constitute more than 70% of total proteins secreted by the fungus. However, their physiological functions and effects on enzymatic hydrolysis of cellulose substrates are not sufficiently elucidated. Here, the cellobiohydrolase-encoding genes cbh1 and cbh2 were deleted, individually or combinatively, by using an auxotrophic marker-recycling technique in T. reesei. When cultured on media with different soluble carbon sources, all three deletion strains (Δcbh1, Δcbh2, and Δcbh1Δcbh2) exhibited no dramatic variation in morphological phenotypes, but their growth rates increased apparently when cultured on soluble cellulase-inducing carbon sources. In addition, Δcbh1 showed dramatically reduced growth and Δcbh1Δcbh2 could hardly grew on microcrystalline cellulose (MCC), whereas all strains grew equally on sodium carboxymethyl cellulose (CMC-Na), suggesting that the influence of the CBHs on growth was carbon source-dependent. Moreover, five representative cellulose substrates were used to analyse the influence of the absence of CBHs on saccharification efficiency. CBH1 deficiency significantly affected the enzymatic hydrolysis rates of various cellulose substrates, where acid pre-treated corn stover (PCS) was influenced the least. CBH2 deficiency reduced the hydrolysis of MCC, PCS, and acid pre-treated and delignified corncob but improved the hydrolysis ability of filter paper. These results demonstrate the specific contributions of CBHs to the hydrolysis of different types of biomass, which could facilitate the development of tailor-made strains with highly efficient hydrolysis enzymes for certain biomass types in the biofuel industry.

[PROTOCOL] Structural analysis of N-/O-glycans assembled on proteins in yeasts: Eun Jung Thak , Jungho Kim , Dong-Jik Lee , Jeong Yoon Kim , Hyun Ah Kang; J. Microbiol. 2018;56(1):11-23. Published online January 4, 2018; DOI: https://doi.org/10.1007/s12275-018-7468-x

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20 Citations

Abstract: Protein glycosylation, the most universal and diverse posttranslational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species- and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N- and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.

Antagonistic effect of peptidoglycan of Streptococcus sanguinis on lipopolysaccharide of major periodontal pathogens: Sung-Hoon Lee; J. Microbiol. 2015;53(8):553-560. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5319-6

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16 Citations

Abstract: Streptococcus sanguinis is often found in subgingival biofilm including periodontopathogens, and is correlated with a delay in colonization by periodontopathogens. However, the effect of S. sanguinis on inflammation induced by periodontopathogens is poorly understood. Thus, this study investigated the effect of S. sanguinis peptidoglycan (PGN) on induction of TNF-α, IL-6, and IL-8 expression by lipopolysaccharide (LPS) of periodontal pathogens. LPS was extracted from Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia, and PGN was isolated from S. sanguinis. THP-1 cells, a monocytic cell-line, were cotreated with LPS of the periodontal pathogens and S. sanguinis PGN, and then the expression of inflammatory cytokines was analyzed by real-time RT-PCR. To analyze the underlying mechanism, the binding assay of the LPS to CD14 or LPS-binding protein (LBP) was performed in the presence or absence of the PGN after coating recombinant human CD14 and LBP on EIA plate. The PGN inhibited the binding of LPS to CD14 and LBP in a dose-dependent manner. Also, THP-1 cells were co-treated with the LPS in the presence of N-acetylmuramic acid and N-acetylglucosamine, as components of PGN, and the competition binding assay to CD14 and LBP was performed. N-acetylmuramic acid inhibited the induction of inflammatory cytokine expression by LPS and the binding of LPS to CD14 or LBP whereas Nacetylglucosamine did not show such effect. Collectively, the
results
suggest that S. sanguinis PGN inhibited the cytokine expression induced by the LPS of periodontopathogens due to the inhibition of LPS binding to LBP and CD14. N-acetylmuramic acid of PGN may play a role in inhibition of the LPS binding of periodontopathogens to CD14 and LBP.

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

Remodeling of the Glycosylation Pathway in the Methylotrophic Yeast Hansenula polymorpha to Produce Human Hybrid-Type N-Glycans: Seon Ah Cheon , Hyunah Kim , Doo-Byoung Oh , Ohsuk Kwon , Hyun Ah Kang; J. Microbiol. 2012;50(2):341-348. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-2097-2

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35 Citations

Abstract: As a step forward to achieve the generation of human complex- type N-glycans in the methylotrophic yeast Hansenula polymorpha, we here report the modification of the yeast glycosylation pathway by heterologous expression of the human gene encoding β-1,2-N-acetylglucosaminyltransferase I (GnTI). For the optimal expression of human GnTI in the yeast Golgi compartment, the catalytic domain of the GnTI was fused to various N-terminal leader sequences derived from the yeast type II membrane proteins. The vectors containing GnTI fusion constructs were introduced into the H. polymorpha och1Δ single and och1Δalg3Δ double mutant strains expressing the ER-targeted Aspergillus saitoi α-1,2 mannosidase, respectively. Both of the glycoengineered Hpoch1Δ and Hpoch1ΔHpalg3Δ strains were shown to produce successfully the hybrid-type glycans with a monoantennary N-acetylglucosamine (GlcNAc1Man5GlcNAc2 and GlcNAc1Man3GlcNAc2, respectively) by N-glycan profile analysis of cell wall proteins. Furthermore, by comparative analysis of byproduct formation and the glycosylation site occupancy, we propose that the Hpoch1Δ strain would be more suitable than the Hpoch1ΔHpalg3Δ strain as a host for the production of recombinant proteins with humanized glycans.

Acinetobacter baumannii Outer Membrane Protein A Modulates the Biogenesis of Outer Membrane Vesicles: Dong Chan Moon , Chul Hee Choi , Jung Hwa Lee , Chi-Won Choi , Hye-Yeon Kim , Jeong Soon Park , Seung Il Kim , Je Chul Lee; J. Microbiol. 2012;50(1):155-160. Published online February 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1589-4

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102 Citations

Abstract: Acinetobacter baumannii secretes outer membrane vesicles (OMVs) during both in vitro and in vivo growth, but the biogenesis mechanism by which A. baumannii produces OMVs remains undefined. Outer membrane protein A of A. baumannii (AbOmpA) is a major protein in the outer membrane and the C-terminus of AbOmpA interacts with diaminopimelate of peptidoglycan. This study investigated the role of AbOmpA in the biogenesis of A. baumannii OMVs. Quantitative and qualitative approaches were used to analyze OMV biogenesis in A. baumannii ATCC 19606T and an isogenic ΔAbOmpA mutant. OMV production was significantly increased in the ΔAbOmpA mutant compared to wild-type bacteria as demonstrated by quantitation of proteins and lipopolysaccharides (LPS) packaged in OMVs. LPS profiles prepared from OMVs from wild-type bacteria and the ΔAbOmpA mutant had identical patterns, but proteomic analysis showed different protein constituents in OMVs from wild-type bacteria compared to the ΔAbOmpA mutant. In conclusion, AbOmpA influences OMV biogenesis by controlling OMV production and protein composition.

Staphylococcal methicillin resistance expression under various growth conditions: Lee, Yoo Nik , Poo Ha Ryoung , Lee, Young Ik; J. Microbiol. 1997;35(2):103-108.

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Abstract: To improve the detection of methicillin resistant staphylococci, lowered incubation temperature (30℃) and inclusion of sodium chloride in media have been empirically recommended. However, in this study, we found that sodium chloride in Peptone-Yeast Extract-K₂HPO₄(PYK) medium decreased methicillin minimum inhibitory concentrations. Divalent cations were shown to restore the expression of staphylococcal methicillin resistance. However, when it was determined by efficiency of plating, sodium chloride increased methicillin resistance expression on agar medium in which higher divalent cations were contained in the agar medium. The decrease of minimum inhibitory concentrations at 30℃ by sodium chloride occurred in Brain Heart Infusion but did not occur in other media investigated. Interestingly, both PYK and Brain Heart Infusion media had peptone, which contain cholic acids having detergent activities. Inclusion of sodium chloride in PYK caused a higher rate of autolysis. Penicillin binding protein 2a that has a low affinity to beta-lactam antibiotics, was highly inducible in methicillin resistant Staphylococcus epidermidis strains. In this study, we found that autolysins that are activated by the sodium chloride decreased the minimum inhibitory concentration at 30℃, and peptidoglycan is weakened due to the presence of methicillin. Peptone in the media may aggravate the fragile cells. However, stabilization due to the presence of divalent cations and production of penicilin binding protein 2a increase the survival of staphylococci.

Intracellular Posttranslational Modification of Aspartyl Proteinase of Candida albicans and the Role of the Glycan Region of the Enzyme: Byoung-Kuk Na , Chul-Yong Song; J. Microbiol. 2000;38(4):218-223.

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Abstract: Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAP1) of Candida albicans. Three intracellular forms of SAP1 were detected by immunoblotting using monoclonal antibody (MAb) CAP1. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAP1 and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAP1. These results show that SAP1 is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor form undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretory vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAP1 was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in part, be related to enzyme stability and activity.

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