Journal of Microbiology

Journal Articles

Functional analysis of ascP in Aeromonas veronii TH0426 reveals a key role in the regulation of virulence: Yongchao Guan , Meng Zhang , Yingda Wang , Zhongzhuo Liu , Zelin Zhao , Hong Wang , Dingjie An , Aidong Qian , Yuanhuan Kang , Wuwen Sun , Xiaofeng Shan; J. Microbiol. 2022;60(12):1153-1161. Published online November 10, 2022; DOI: https://doi.org/10.1007/s12275-022-2373-8

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Abstract: Aeromonas veronii is a pathogen which can induce diseases in humans, animals and aquatic organisms, but its pathogenic mechanism and virulence factors are still elusive. In this study, we successfully constructed a mutant strain (ΔascP) by homologous recombination. The results showed that the deletion of the ascP gene significantly down-regulated the expression of associated effector proteins in A. veronii compared to its wild type. The adhesive and invasive abilities of ΔascP to EPC cells were 0.82-fold lower in contrast to the wild strain. The toxicity of ΔascP to cells was decreased by about 2.91-fold (1 h) and 1.74-fold (2 h). Furthermore, the LD50 of the mutant strain of crucian carp was reduced by 19.94-fold, and the virulence was considerably attenuated. In contrast to the wild strain, the ΔascP content in the liver and spleen was considerably lower. The titers of serum cytokines (IL-8, TNF-α, and IL-1β) in crucian carp after the infection of the ΔascP strain were considerably lower in contrast to the wild strain. Hence, the ascP gene is essential for the etiopathogenesis of A. veronii TH0426.

Mediterraneibacter butyricigenes sp. nov., a butyrate-producing bacterium isolated from human faeces: Ji-Sun Kim , Keun Chul Lee , Min Kuk Suh , Kook-Il Han , Mi Kyung Eom , Ju Huck Lee , Seung-Hwan Park , Se Won Kang , Jam-Eon Park , Byeong Seob Oh , Seung Yeob Yu , Seung-Hyeon Choi , Dong Ho Lee , Hyuk Yoon , Byung-Yong Kim , Seung-Jo Yang , Jung-Sook Lee; J. Microbiol. 2019;57(1):38-44. Published online December 29, 2018; DOI: https://doi.org/10.1007/s12275-019-8550-8

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Abstract: A Gram-stain-positive, obligately anaerobic, non-motile, nonspore- forming, and rod-shaped bacterial strain, designated KGMB01110T, was isolated from a faecal sample of a healthy male in South Korea. Phylogenetic analysis based on 16S rRNA gene showed that strain KGMB01110T belonged to Clostridium cluster XIVa and was most closely related to Mediterraneibacter glycyrrhizinilyticus KCTC 5760T (95.9% 16S rRNA gene sequence similarity). The DNA G + C content of strain KGMB01110T based on its whole genome sequence was 44.1 mol%. The major cellular fatty acids (> 10%) of the isolate were C14:0 and C16:0. The strain KGMB01110T was positive for arginine dihydrolase, β-galactosidase-6-phosphatase, and alkaline phosphatase. The strain KGMB01110T also produced acid from D-glucose and D-rhamnose, and hydrolyzed gelatin and aesculin. Furthermore, HPLC analysis and UV-tests of culture supernatant revealed that the strain KGMB01110T produced butyrate as the major end product of glucose fermentation. Based on the phylogenetic and phenotypic characteristics, strain KGMB01110T represent a novel species of the genus Mediterraneibacter in the family Lachnospiraceae. The type strain is KGMB01110T (= KCTC 15684T = CCUG 72830T).

Cultivable butyrate-producing bacteria of elderly Japanese diagnosed with Alzheimer’s disease: Thi Thuy Tien Nguyen , Yuta Fujimura , Iyo Mimura , Yusuke Fujii , Ngoc Luong Nguyen , Kensuke Arakawa , Hidetoshi Morita; J. Microbiol. 2018;56(10):760-771. Published online August 22, 2018; DOI: https://doi.org/10.1007/s12275-018-8297-7

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Abstract: The group of butyrate-producing bacteria within the human gut microbiome may be associated with positive effects on memory improvement, according to previous studies on dementia- associated diseases. Here, fecal samples of four elderly Japanese diagnosed with Alzheimer’s disease (AD) were used to isolate butyrate-producing bacteria. 226 isolates were randomly picked, their 16S rRNA genes were sequenced, and assigned into sixty OTUs (operational taxonomic units) based on BLASTn results. Four isolates with less than 97% homology to known sequences were considered as unique OTUs of potentially butyrate-producing bacteria. In addition, 12 potential butyrate-producing isolates were selected from the remaining 56 OTUs based on scan-searching against the PubMed and the ScienceDirect databases. Those belonged to the phylum Bacteroidetes and to the clostridial clusters I, IV, XI, XV, XIVa within the phylum Firmicutes. 15 out of the 16 isolates were indeed able to produce butyrate in culture as determined by high-performance liquid chromatography with UV detection. Furthermore, encoding genes for butyrate formation in these bacteria were identified by sequencing of degenerately primed PCR products and included the genes for butyrate kinase (buk), butyryl-CoA: acetate CoAtransferase (but), CoA-transferase-related, and propionate CoA-transferase. The results showed that eight isolates possessed buk, while five isolates possessed but. The CoA-transfer- related gene was identified as butyryl-CoA:4-hydroxybutyrate CoA transferase (4-hbt) in four strains. No strains contained the propionate CoA-transferase gene. The biochemical and butyrate-producing pathways analyses of butyrate producers presented in this study may help to characterize the butyrate-producing bacterial community in the gut of AD patients.

Effects of dietary poly-β-hydroxybutyrate (PHB) on microbiota composition and the mTOR signaling pathway in the intestines of Litopenaeus vannamei: Yafei Duan , Yue Zhang , Hongbiao Dong , Yun Wang , Jiasong Zhang; J. Microbiol. 2017;55(12):946-954. Published online December 7, 2017; DOI: https://doi.org/10.1007/s12275-017-7273-y

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47 Citations

Abstract: Poly-β-hydroxybutyrate (PHB) is a natural polymer of the short chain fatty acid β-hydroxybutyrate, which acts as a microbial control agent. The mammalian target of the rapamycin (mTOR) signaling pathway plays a crucial role in intestine inflammation and epithelial morphogenesis. In this study, we examined the composition of intestine microbiota, and mTOR signaling-related gene expression in Pacific white shrimp Litopenaeus vannamei fed diets containing different levels of PHB: 0% (Control), 1% (PHB1), 3% (PHB3), and 5% (PHB5) (w/w) for 35 days. High-throughput sequencing analysis revealed that dietary PHB altered the composition and diversity of intestine microbiota, and that the microbiota diversity decreased with the increasing doses of PHB. Specifically, dietary PHB increased the relative abundance of Proteobacteria and Tenericutes in the PHB1 and PHB5 groups, respectively, and increased that of Gammaproteobacteria in the three PHB groups. Alternatively, PHB decreased Alphaproteobacteria in the PHB3 and PHB5 groups. At the genus level, dietary PHB increased the abundance of beneficial bacteria, such as Bacillus, Lactobacillus, Lactococcus, Clostridium, and Bdellovibrio. The relative mRNA expression levels of the mTOR signaling-related genes TOR, 4E-BP, eIF4E1α, and eIF4E2 all increased in the three PHB treatment groups. These
results
revealed that dietary PHB supplementation had a beneficial effect on intestine health of L. vannamei by modulating the composition of intestine microbiota and activating mTOR signaling.

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

NOTE] Biosynthesis of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Copolyesters with a High Molar Fraction of 3-Hydroxyvalerate by an Insect-Symbiotic Burkholderia sp. IS-01: Do Young Kim , Doo-Sang Park , Soon Bum Kwon , Moon Gyu Chung , Kyung Sook Bae , Ho-Yong Park , Young Ha Rhee; J. Microbiol. 2009;47(5):651-656. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0109-7

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Abstract: Burkholderia sp. IS-01 capable of biosynthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB- co-3HV)] copolyesters with a high molar fraction of 3HV was isolated from the gut of the adult longicorn beetle, Moechotypa diphysis. The strain IS-01 was relatively tolerant to high concentrations of levulinic acid and accumulated a poly(13.5 mol% 3HB-co-86.5 mol% 3HV) copolyester when cultivated on a mixture of gluconate (20 g/L) and levulinic acid (12.5 g/L). In this case, the content of the copolyester in the cells was approximately 60.0%. The compositions of the copolyesters were easily regulated by altering the molar ratio of gluconate and levulinic acid in the medium. The organism was found to possess a class I PHA synthase (PhaC) gene (1,881 bp) that encodes a protein with a deduced molecular mass of 68,538 Da that consists of 626 amino acids. The PhaC of this organism was most similar to that of B. cenocepacia PC184 (92% similarity).

Stable Expression and Secretion of Polyhydroxybutyrate Depolymerase of Paucimonas lemoignei in Escherichia coli: Se Whan Park , Moon Gyu Chung , Hwa Young Lee , Jeong Yoon Kim , Young Ha Rhee; J. Microbiol. 2008;46(6):662-669. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0283-z

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Abstract: An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of PhaZ1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing PhaZ1 and its own N-terminal signal peptide (PrePhaZ1) enabled the secretion of active PhaZ1 into the extracellular medium. However, the PrePhaZ1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-PhaZ1 and INPNCPrePhaZ1 fusion constructs did not affect growth of host cells. INPNC-PhaZ1 was successfully displayed on the cell surface with its fusion form, but did not retain PhaZ1 activity. In the case of INPNC-PrePhaZ1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active PhaZ1 was consequently released into the culture medium. The amount of PhaZ1 derived from E. coli (INPNC-PrePhaZ1) was almost twice as great as that directly expressed from E. coli (PrePhaZ1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22°C but was efficiently secreted into the extracellular medium when cultivated at 37°C.

Isolation of Glucose Utilizing Mutant of Alcaligenes eutrophus, its Substrate Selectivity, and Accumulation of Poly-β-hydroxybutyrate: Kim, Hye Yeon , Park, Jin Seo , Shin, Hyun Dong , Lee, yong Hyun; J. Microbiol. 1995;33(1):51-58.

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Abstract: A glucose utilizing mutant was selected from parent strain Alcaligenes eutrophus H16, and named as Glu-9. The mechanisms of glucose utilization of the mutant Glu-9 was investigated by measuring the D-[1-¹⁴C] glucose transport activity and the activities of key enzymes related to glucose and fructose uptake via facilitated diffusion. The uptaken glucose seems to activate key enzymes related to glucose matabolism. The selectivity between glucose and fructose of mutant Glu-9 was also analyzed by measuring glucose transport activity and enzyme activities under the various cultivation conditions using different carbon sources. Mutant Alcaligenes eutrophus Glu-9 preferentially consumed fructose from mixed substrates of glucose and fructose due to the inhibition of fructose to glucose transport activity. The characteristics of cell growth and PHB accumulation of Alcaligenes eutrophus Glu-9 were examined under various cultural conditions. Mutant strain Glu-9 showed tolerance in high concentration of glucose and increased yield of PHB production.

Biosynthesis of Poly-β-Hydrozyalkanoates by Bacillus thuringiensis R-510: Lee, Kang Tae , Kim, Jeong Yoon , Rhee, young Ha , Bae, Kyung Sook , Kim, Young Baek; J. Microbiol. 1995;33(1):59-65.

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Abstract: Synthesis and accumulation of Poly-β-Hydrozyalkanoates (PHA) in Bacillus thuringiensis R-510 isolated from soil were investigatd. This organism was resistant to relatively higj concentration of propionate and had a capability of accumulatinf copolymers consisting of 3-hydroxybutyrate(3HB) and 3-hydroxyvalerate(3HV) when the medium was supplemented with propionate as a precursor, The PHA content maximally reached up to 44.5% of dry cell weight in the presence of 0.1% propionate. The molar fraction of 3HV in the copolymer was increased from 19.4 to 80,2 mol% by adding 0.05 to 0.5% propionate to glucose medium. The addition of propionate during exponential or stationary phase of cell growth was less effective for the enhancement of 3HV content in the copolymer, although cell mass and PHA content were not affected by the time of propionate addition. PHB homopolymer and copolymer produced by B. thuringiensis R-510 were measured to have number average molecular weights in the range of 53,000 to 65,000. Polydispersity indices were between 1.5 and 2.2. Some of the produced polymers had bimodal molecular weight distribution.

Biosynthesis of polyhydroxybutyrate and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacillus thuringiensis R-510: Park, Sang Kyu , Lee, Kang Tae , Kim, Young Baek , Rhee, Young Ha; J. Microbiol. 1997;35(2):127-133.

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Abstract: Biosynthesis of polyhydroxybutyrate and copolymer consisting of 3-hydroxybutyrate and 3-hydroxyvalerate [poly(3HB-co-3HV)] by Bacillus thuringiensis R-510 grown with glucose or with mixtures of glucose and propionate was investigated. n-Alkanoic acids other than propionate were not precursors of 3HV units. The fraction of 3HV unit in the copolymer increased from 0 to 84 mol% of 3HV. Polymer yield decreased as the fraction of propionate was increased but the molecular weight distribution was not affected by the composition of carbon substrate. The minimum melting temperature (around 65℃) of poly (3HB-co-3HV) copolymers was observed for the polymer bearing approximately 35 mol% of 3HV. Polyhydroxyalkanoates production by this organism was not dependent on nutritional limitation, but remarkably influenced by dissolved oxygen concentration in the culture medium. Low level of dissolved oxygen concentration prevented spore formation in the cells and stimulated the synthesis of polyhydroxyalkanoate. The composition of poly (3HB-co-3HV) produced by B. thuringiensis R-510 lyhydroxyalkanoate. The composition of poly(3HB-co-3HV) produced by B. thuringiensis R-510 varied according to the growth time. However, there was no evidence that polymers isolated from cells were mixtures of immiscible polymers.

Purification and Properties of Extracellular Poly(3-hydroxybutyrate) Depolymerase Produced by Penicillium pinophilum: Han, Jeen Sun , Son, Young Jong , Chang, Chung Soon , Kim, Mal Nam; J. Microbiol. 1998;36(2):67-73.

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Abstract: The extracellular poly(3-hydroxybutyrate)(PHB) depolymerase of Penicilliyum pinophilum ATCC 9644 was purified and characterized. When Penicillum sp. was grown in basal salt medium with PHB as a sole carbon source, higher temperature favored fungal mycelial growth (37℃>30℃>25℃), but enzyme production was lower ar 37℃ than at any other temperatures. The PHB depolymerase was purified using Sepharose CL06B and Sephacryl S0100HR column chromatography. The isolated enzyme was found to be composed of a single polypeptide chain with a molecular weight of about 35 kDa. The optimum condition for the enzyme was pH 6.0 and 50℃, Enzyme activity decreased sharply at temperatures above 50℃. The enzyme was found to be stable in the pH range of 2.0~10.0.1mM Fe^2+ reduced the enzyme activity by 55% abd 4 mM Fe^2+ inhibited it almost completely. The PHB depolymerase (10U) degraded 47% of solvent cast PHB film, while commercial lipase (1000U) of Rhizopus arrhizus degraded 10% of the same specimen, over a period of 48 hours.

Purification and Characterization of Extracellular Poly(3-hydroxybutyrate) Depolymerase from Penicillium simplicissimum LAR13: Jee-Sun Han , Mal-Nam Kim; J. Microbiol. 2002;40(1):20-25.

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Abstract: An extracellular PHB depolymerase was purified from P. simplicissimum LAR13 cultural medium by Sepharose CL-6B chromatography. When the fungus was grown in a basal salt medium with poly(3-hydroxybutyrate) (PHB) as the sole carbon source, PHB depolymerase production reached maximum at its stationary phase. The mycelial growth rate was higher at 37 C than at 30 C and even higher than at 25 C, However, the enzyme production was lower at 37 C than 30 C or 25 C. The isolated enzyme is composed of a single polypeptide chain with a molecular mass of about 36 kDa as determined by SDS-PAGE. The optimum conditions for the enzyme activity are pH 5.0 and 45 C. The enzyme was stable for 30 min at a temperature lower than 50 C, and stable at pH higher than 2.0 but it was unstable at pH 1.0. 1 mM Fe^2+ reduced the enzyme activity by 56% and the enzyme was inhibited almost completely by 4 mM Fe^2+ . The enzyme was partially inhibited by phenylmethylsulfonyl fluoride and was very sensitive to diazo-DL-norleucine methyl ester, dithiothreitol and mercuric ion. However, N-p-tosyl-L-lysinechloromethyl ketone, p-hydroxymercuricbenzoate and N-acetylimidazole had no influence upon its activity.

Purification and Characterization of Poly(3-hydroxybutyrate) Depolymerase from a Fungal Isolate, Emericellopsis minima W2: Do Young Kim , Ji Hye Yun , Hyung Woo Kim , Kyung Sook Bae , Young Ha Rhee; J. Microbiol. 2002;40(2):129-133.

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Abstract: The fungus, Emericellopsis minima W2, capable of degrading poly(3-hydroxybutyrate) (PHB) was isolated from a waste water sample. Production of the PHB depolymerase from E. minima W2 (PhaZ_Emi ) was significantly repressed in the presence of glucose. PhaZ_Emi was purified by column chromatography on Octyl-Sepharose CL-4B and Sephadex G-100. The molecular mass of the PhaZ_Emi , which consisted of a single polypeptide chain, was estimated to be 48.0 kDa by SDS-PAGE and its pI value was 4.4. The maximum activity of the PhaZ_Emi was observed at pH 9.0 and 55 C. It was significantly inactivated by 1 mM dithiothreitol, 2 mM diisopropyl fluorophosphate, 0.1 mM Tween 80, and 0.1 mM Triton X-100, but insensitive to phenylmethylsulfonyl fluoride and N-ethylmaleimide. The PhaZ_Emi efficiently hydrolyzed PHB and its copolyester with 30 mol% 3-hydroxyvalerate, but did not act on poly(3-hydroxyoctanoate). It also hydrolyzed p-nitrophenylacetate and p-nitrophenylbutyrate but hardly affected the longer-chain forms. The main hydrolysis product of PHB was identified as a dimer of 3-hydroxybutyrate.

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