Journal of Microbiology

Review

Structural Insights into the Lipopolysaccharide Transport (Lpt) System as a Novel Antibiotic Target: Yurim Yoon, Saemee Song; J. Microbiol. 2024;62(4):261-275. Published online May 31, 2024; DOI: https://doi.org/10.1007/s12275-024-00137-w

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Abstract: Lipopolysaccharide (LPS) is a critical component of the extracellular leaflet within the bacterial outer membrane, forming an effective physical barrier against environmental threats in Gram-negative bacteria. After LPS is synthesized and matured in the bacterial cytoplasm and the inner membrane (IM), LPS is inserted into the outer membrane (OM) through the ATP-driven LPS transport (Lpt) pathway, which is an energy-intensive process. A trans-envelope complex that contains seven Lpt proteins (LptA-LptG) is crucial for extracting LPS from the IM and transporting it across the periplasm to the OM. The last step in LPS transport involves the mediation of the LptDE complex, facilitating the insertion of LPS into the outer leaflet of the OM. As the Lpt system plays an essential role in maintaining the impermeability of the OM via LPS decoration, the interactions between these interconnected subunits, which are meticulously regulated, may be potential targets for the development of new antibiotics to combat multidrug-resistant Gram-negative bacteria. In this review, we aimed to provide an overview of current research concerning the structural interactions within the Lpt system and their implications to clarify the function and regulation of LPS transport in the overall process of OM biogenesis. Additionally, we explored studies on the development of therapeutic inhibitors of LPS transport, the factors that limit success, and future prospects.

Journal Article

PROTOCOL] Applications of different solvents and conditions for differential extraction of lipopolysaccharide in Gram-negative bacteria: Mai Phuong Nguyen , Le Viet Ha Tran , Hyun Namgoong , Yong-Hak Kim; J. Microbiol. 2019;57(8):644-654. Published online May 23, 2019; DOI: https://doi.org/10.1007/s12275-019-9116-5

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Lipopolysaccharide (LPS) is one of the major components in the outer membrane of Gram-negative bacteria. However, its heterogeneity and variability in different bacteria and differentiation conditions make it difficult to extract all of the structural variants. We designed a solution to improve quality and biological activity of LPS extracted from various bacteria with different types of LPS, as compared to conventional
methods
. We introduced a quality index as a simple measure of LPS purity in terms of a degree of polysaccharide content detected by absorbance at 204 nm. Further experiments using gel electrophoresis, endotoxin test, and macrophage activation test were performed to evaluate the performance and reliability of a proposed ‘T-sol’ method and the biological effectiveness and character of the LPS products. We presented that the T-sol method had differential effects on extraction of a RAW 264.7 cell-activating LPS, which was effective in the macrophage activation with similar effects in stimulating the production of TNF-alpha. In conclusion, the T-sol method provides a simple way to improve quality and biological activity of LPS with high yield.

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Effective Modalities of Periodontitis Induction in Rat Model
Fazle Khuda, Badiah Baharin, Nur Najmi Mohamad Anuar, Bellen Sharon Fred Satimin, Nurrul Shaqinah Nasruddin
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Review

MINIREVIEW] Two stress sensor proteins for the expression of sigmaE regulon: DegS and RseB: Dong Young Kim; J. Microbiol. 2015;53(5):306-310. Published online May 3, 2015; DOI: https://doi.org/10.1007/s12275-015-5112-6

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Abstract

In E. coli, sigmaE-dependent transcription is controlled by regulated-proteolysis of RseA. RseA, which holds sigmaE as an anti-sigma factor, is sequentially digested by DegS, RseP and cytoplasmic proteases to liberate sigmaE in response to dysfunction in outer-membrane biogenesis. Additionally, the sequential proteolysis is regulated by RseB binding to RseA (Fig. 1A). Direct interaction between RseA and RseB inhibits RseA-cleavage by DegS. Both proteolytic activation of DegS and binding disruption of RseB are thus required to initiate sigmaE-stress response. For the induction of sigmaEstress response, DegS and RseB recognize the states of OMP and LPS for outer-membrane biogenesis. DegS is activated by binding of unfolded OMPs and RseB binding to RseA is antagonized by LPS accumulated in periplasm. In this regard, DegS and RseB are proposed to be stress sensor proteins for sigmaE signal transduction. Interestingly, biogenesis of OMP and LPS appears to cross-talk with each other, indicating that dysfunction of either OMP or LPS can initiate RseA proteolysis. This review aims to briefly introduce two stress sensor proteins, DegS and RseB, which regulate sigmaEdependent transcription.

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Liang Wang, Hao Yang, Mengping Wu, Jianhua Zhang, Hongjian Zhang, Zhonggui Mao, Xusheng Chen
Frontiers in Microbiology.2023;[Epub] CrossRef
A σE-mediated temperature gauge orchestrates type VI secretion system, biofilm formation and cell invasion in pathogen Pseudomonas plecoglossicida
Yibei Zhang, Yuping Huang, Haoyuan Ding, Jiabao Ma, Xinyu Tong, Yuanxing Zhang, Zhen Tao, Qiyao Wang
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Research Support, Non-U.S. Gov'ts

Extracellular Stress and Lipopolysaccharide Modulate Acinetobacter baumannii Surface-Associated Motility: Christin N. McQueary , Benjamin C. Kirkup , Yuanzheng Si , Miriam Barlow , Luis A. Actis , David W. Craft , Daniel V. Zurawski; J. Microbiol. 2012;50(3):434-443. Published online June 30, 2012; DOI: https://doi.org/10.1007/s12275-012-1555-1

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Abstract: Acinetobacter baumannii is a nosocomial bacterial pathogen, and infections attributed to this species are further complicated by a remarkable ability to acquire antimicrobial resistance genes and to survive in a desiccated state. While the antibiotic resistance and biofilm formation of A. baumannii is well-documented, less is known about the virulence attributes of this organism. Recent studies reported A. baumannii strains display a motility phenotype, which appears to be partially dependent upon Type IV pili, autoinducer molecules, and the response to blue light. In this study, we wanted to determine the prevalence of this trait in genetically diverse clinical isolates, and any additional required factors, and environmental cues that regulate motility. When strains are subjected to a wide array of stress conditions, A. baumannii motility is significantly reduced. In contrast, when extracellular iron is provided or salinity is reduced, motility is significantly enhanced. We further investigated whether the genes required for the production of lipopolysaccharide (lpsB) and K1 capsule (epsA/ptk) are required for motility as demonstrated in other Gram-negative bacteria. Transposon mutagenesis resulted in reduced motility by the insertion derivatives of each of these genes. The presence of the parental allele provided in trans, in the insertion mutant background, could only restore motility in the lpsB mutant. The production of core LPS directly contributes to the motility phenotype, while capsular polysaccharide may have an indirect effect. Further, the data suggest motility is regulated by extracellular conditions, indicating that A. baumannii is actively sensing the environment and responding accordingly.

Mouse Strain-Dependent Osteoclastogenesis in Response to Lipopolysaccharide: Ho Gil Choi , Jin Moon Kim , Bong-Ju Kim , Yun-Jung Yoo , Jeong-Heon Cha; J. Microbiol. 2007;45(6):566-571.; DOI: https://doi.org/2607 [pii]

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Abstract: Bacterial lipopolysaccharide (LPS) is a potent stimulator of bone resorption in periodontitis. Co-culture systems of mouse calvaria-derived osteoblasts and bone marrow-derived preosteoclasts were used as an in vitro osteoclast differentiation. This study revealed that co-cultures using ddY or ICR mouse strain responded differently to LPS while responded equally to 1α,25(OH)2D3. Thus, the different response to LPS indicates dissimilarity of two mouse stains in their capacity for generating osteoclasts while the two mouse strains share the similarity in response to 1α,25(OH)2D3. To identify which cells between osteoblasts and preosteoclasts in the co-culture are responsible for the dissimilarity, the reciprocal co-cultures were performed between ddY and ICR mouse strains. The treatment of 1,25(OH)2D3 to ddY/ICR (osteoblasts from ddY/preosteoclasts from ICR) and ICR/ddY reciprocal co-cultures also showed the similarity. In case of LPS treatment, the results of ddY/ICR were similar to ddY/ddY and the results of the other reciprocal co-culture, ICR/ddY combination, were consistent with those of ICR/ICR. It suggests that the dissimilarity between the two mouse strains may resident in osteoblasts but not in preosteoclasts. Therefore, the osteoblast is responsible for mouse strain-dependent osteoclastogenesis in response to LPS. Although mouse models will continue to provide insights into molecular mechanisms of osteoclastogenesis, caution should be exercised when using different mouse strains, especially ddY and ICR strains as models for osteoclast differentiation.

Independent regulation of antigen processing and presentation on induction of antibody responses to various bacterial antigens in C3H/He mice: Kim, Hyung Su , Jeong, Ga Jin; J. Microbiol. 1995;33(4):355-362.

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Abstract: Induction of antibody production in C3H/He mice by bacterial infection is regulated through the processing exerted by antigen presenting cells. From the studies with Psudomonas aeruginosa, Salmonella typhimurium, and Micrococcus luteu, lipopolysaccharides (LPS) in Gram negative bacteria, which are known to be T-cell independent B cell mitogen, seem to be the major factor stimulating immune responses via activation of macrophages. Activation of macrophage, however, does not seem to correlate with antibody production. M. luteus was easily eliminatd by activated macrophages, while the processed antigens were immediately releasedd into culture medium before presentation. Nevertheless, antigens from Gram positive bacteria, Staphylococcus aureus and Bacillus subtilis, were very very active in chemotaxis and activation of periotoneal macrophages as well as in antien presnetation, while the very nature of the antigens is not yet clearly understood.

Phylogenetic relationship of ganoderma species with the polyporaceae base don RFLP analysis of the nuclear ITS region: Park, Hong Je , Shin, Kee Sun , Yoon, Cheol Sik , Lee, Dong Hun , Bae, Kyung Sook; J. Microbiol. 1996;34(2):117-123.

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Abstract: Restriction-polymorphic patterns of nuclear-ITS were examined for the genetic relationships among 12 bisidiomycetous mushrooms to Aphyllophorales and Agaricales. The taxonomic affinity of Ganoderma species with the family Polyporaceae also was examined. With 13 restriction endonucleases, 159 restriction characters were generated form the 12 species examined. UPGMA and neighbor-joining analyses separated the 12 species into two genetically distinct groups that correspond to orders (Agaricales and Aphyllophorales) where each species is included. This result indicates that there is clear genetic demarcation between Agaricales and Aphyllophorales. Dendrograms constructed by several data analyses showed that even though Ganoderma species are somewhat in intermediate taxonomic position between the Polyporaceae and families of the Agaricales, they are genetically more related to the Polyporaceae. These results are consistent with morphological characters observed in those mushrooms. However, it is premature to conclude taxonomic status Ganoderma species in the present study employing small sample size.

Phylogenetic study of trichaptum species based on the RFLP analysis of mitochondrial DNA: Kim, Mi Sun , jung, Hack Sung; J. Microbiol. 1996;34(3):215-219.

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Abstract: Eight strains of Trichaptunm (Polyporaceae), two strains from each species of T. abietinum, T. biforme, T. fusco-violaceum, and T. laricinum were examined to see their phylogenetic relationship by digesting mitochondrial DNAs with EcoRV, Hind III, XbaI, and PstI, and then analyzing fragmentation patterns with the methods of Nei and Li. T. abietinum, T. biforme, and T. laricinum developed an independent phylogenetic lineage, respectively, but T. fusco-violaceum FP-133997-sp showed a close relationship with two strains of T. bioforme, and T. fusco-violaceum HHB-4016-sp barely grouped with those of T. laricinum. Based on the results of the RFLP analysis of mtDNA, it is concluded that T. fusco-violaceum is under way to differentiation into two different subgroups.

Lipoppolysaccharide yields form Rhodobacter capasulatus with indirect ELISA: Yoo, Tae Eun , Lee, Hyun Soon; J. Microbiol. 1996;34(3):255-262.

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Abstract: The lipopolysaccharide (LPS) yields were measured in Rhodobacter capsulatus under several conditions by the ELISA method. The purification of LPS was done by affinity chromatography of IgG coupled CNBr-activated sepharose-4B instead of ultra-centrifugation. The purity of the LPS didn't show much difference between affinity chromatography and ultra-centrifugation method, but affinity chromatography method required much fewer organisms and was more convenient. LPS yield was measured in ng units by the ELISA method. Mannitol was a better single carbon source than other sugars, but mixing two carbon sources resulted in greater LPS yields than any sugar alone. LPS yield was directly proportional to NH₄CI concentration, with optimum yields at 0.05% nitrogen. In contrest to LPS yields, which decreased at 0.005% nitrogen concentration total protein was increased 16 times. Calcium influenced LPS yields. At 0.7 mM CaCI₂, the LPS yield was 16.5 ㎍/mg DW, five times the yield without calcium.

A Ser/Thr Specific Protein Kinase Activates the Mouse Rantes Gene after Lipopolysaccharide Stimulation: Youn- Uck Kim , Youn-Hwoan Kim , Duek-Jun An , Hyuk-Chu Kwon; J. Microbiol. 2001;39(4):314-320.

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Abstract: Macrophages stimulated by lipopolysaccharide (LPS) from gram negative bacteria undergo activation of a group of immediate early genes including Rantes. The mouse Rantes gene promoter region contains an LPS responsive element (LRE). We detected 3 specific bands, termed B1, B2, and B3, formed by the interaction of the LRE and proteins found in LPS-stimulated RAW 264.7 cells. An additional band, B4, was determined to be an AP-1 binding protein. The B1 band appears within 1 hour of LPS stimulation. The observed binding pattern could be changed by a specific heparin column fraction of nuclear extracts from LPS-stimulated cells. We have determined that a Ser/Thr-specific protein kinase is activated by LPS stimulation, and this protein kinase enhances B1 band formation. The B1 band can be converted to band B2/B3 by adding specific heparin column fraction. Purified Ser/Thr-specific protein phosphatases PP-1 and PP-2A can stimulate the same conversion to about the same extent. Thus, the formation of the LRE sequence binding complex appears to be regulated by Ser/Thr protein kinase and one or more Ser/Thr specific phosphatases. At least four proteins are involved in the regulation of the LRE-dependent Rantes expression: two binding factors that bind directly to the target sequences, and two factors that control their binding. The future purification and characterization of these binding proteins will reveal in detail the mechanism of Rantes gene activation after LPS stimulation.

PCR-DGGE and PCR-RFLP Analyses of the Internal Transcribed Spacer (ITS) of Ribosomal DNA in the Genus Rhizopus: You-Jung Park , Yong-Keel Choi , Byung-Re Min; J. Microbiol. 2003;41(2):157-160.

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Abstract: To estimate genetic relationships within the genus Rhizopus, genetic variations in 20 strains were investigated by DGGE and PCR-RFLP of rDNA ITS region (ITS1, ITS2, 5.8S). The size of the amplified products showed the interspecific polymorphisms, 650 bp, 700 bp, and 900 bp. The DGGE approach allowed the separation of PCR amplicons of the same length according to their sequence variations. When the rDNA ITS region was digested with six restriction enzymes, 20 strains were classified into five RFLP haplotypes. The range of similarity between the 20 strains by PCR-RFLP was 42.3-100%. Based on the results of DGGE and PCR-RFLP, the 20 strains were divided into four groups, R. oryzae, R. stolonifer, R. microsporus and R. homothallicus. Further study of R. homothallicus is required.

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