Journal of Microbiology

Volume 58(9); September 2020

Review

[MINIREVIEW]Phosphate sugar isomerases and their potential for rare sugar bioconversion: Soo-Jung Kim , Yeong-Su Kim , Soo-Jin Yeom; J. Microbiol. 2020;58(9):725-733. Published online June 25, 2020; DOI: https://doi.org/10.1007/s12275-020-0226-x

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Abstract: Phosphate sugar isomerases, catalyzing the isomerization between ketopentose/ketohexose phosphate and aldopentose/ aldohexose phosphate, play an important role in microbial sugar metabolism. They are present in a wide range of microorganisms. They have attracted increasing research interest because of their broad substrate specificity and great potential in the enzymatic production of various rare sugars. Here, the enzymatic properties of various phosphate sugar isomerases are reviewed in terms of their substrate specificities and their applications in the production of valuable rare sugars because of their functions such as low-calorie sweeteners, bulking agents, and pharmaceutical precursor. Specifically, we focused on the industrial applications of D-ribose-5-phosphate isomerase and D-mannose-6-phosphate isomerase to produce D-allose and L-ribose, respectively.

Journal Articles

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).

Changes in the microbial community of Litopenaeus vannamei larvae and rearing water during different growth stages after disinfection treatment of hatchery water: Yafei Duan , Yapeng Tang , Jianhua Huang , Jiasong Zhang , Heizhao Lin , Shigui Jiang , Ruixuan Wang , Guofu Wang; J. Microbiol. 2020;58(9):741-749. Published online July 24, 2020; DOI: https://doi.org/10.1007/s12275-020-0053-0

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Abstract: Microbial communities greatly affect rearing water quality and the larvae health during shrimp hatchery periods. In this study, we investigated the microbial communities of rearing water and larvae of Litopenaeus vannamei after treating hatchery water with different kinds of chemical disinfectants: no disinfectants (Con), chlorine dioxide (ClO2), formaldehyde solution (HCHO), bleach powder (CaClO), and iodine (I2). The water and larval samples were collected from nauplius 6 (N6), zoea 1 (Z1), mysis 1 (M1), and postlarvae 1 (P1) shrimp growth periods. 16S rDNA high-throughput sequencing revealed that the bacterial composition of the rearing water was more complex than that of the larvae, and the bacterial community of the rearing water and the larvae fluctuated significantly at the P1 and Z1 periods, respectively. Disinfectants altered the bacterial diversity and composition of the rearing water and larvae. Specifically, in the rearing water of the P1 period, Proteobacteria abundance was increased in the HCHO group; while Bacteroidetes abundance was decreased in the ClO2, HCHO, and I2 groups but increased in the CaClO group. In the larvae of the Z1 period, Firmicutes (especially Bacillus class) abundance was increased in the CaClO group, but decreased in the ClO2, HCHO, and I2 groups. Network analyses revealed that the genera Donghicola, Roseibacterium, Candidatus-Cquiluna, and Nautella were enriched in the rearing water, while Halomonas, Vibrio, and Flavirhabdus had high abundance in the larvae. The survival of shrimp was influenced by disinfectants that were inconsistent with the bacterial community changes. These results will be helpful for using microbial characteristics to facilitate healthy shrimp nursery.

The relationship between bacterial diversity and organic carbon mineralization in soft rock and sand compound soil: Zhen Guo , Juan Li , Lei Ge , Chenxi Yang , Jichang Han; J. Microbiol. 2020;58(9):750-760. Published online July 24, 2020; DOI: https://doi.org/10.1007/s12275-020-0130-4

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Abstract: The soil organic carbon (SOC) mineralization rate in sandy soil plays an important role in improving soil quality, and a research is needed to determine management practices that optimize the mineralization rate. When sandy soil is improved by adding soft rock, the specific promotion process of bacterium to SOC mineralization remain unclear. To investigate these mechanisms, we selected four treatments with soft rock to sand volume ratios of 0:1 (CK), 1:5 (C1), 1:2 (C2) and 1:1 (C3) to study. The mineralization rate of organic carbon was measured using the lye absorption method. Highthroughput sequencing and scanning electron microscopy were used to determine the bacterial community structure and soil microstructure, respectively. The results showed that the organic carbon content of the sandy soil increased significantly (182.22–276.43%) after using the soft rock treatments. The SOC mineralization rate could be divided into two stages: a rapid decline during days 1–8 and a slow decline during days 8–60. With increased incubation time, the intensity of the cumulative release of organic carbon gradually weakened. Compared with the CK treatment, the SOC mineralization accumulation (Ct) and the potential mineralizable organic carbon content (C0) in the C1, C2, and C3 treatments increased significantly, by 106.98–225.94% and 112.22– 254.08%, respectively. The cumulative mineralization rate (Cr) was 18.11% and 21.38% smaller with treatments C2 and C3, respectively. The SOC mineralization rate constant (k) decreased significantly after the addition of soft rock, while the half-turnover period (Th) changed inversely with k. Compared with the CK treatment, the number of gene copies of the soil bacteria increased by 15.38–272.53% after adding soft rock, with the most significant increase in treatment C3. The bacterial diversity index also increased significantly under treatment C3. The three dominant bacteria were Proteobacteria, Actinobacteria, and Chloroflexi. The correlation between Cr and one of the non-dominant bacteria, Firmicutes, was large, and the bacteria had a significant positive correlation with k. At the same time, the abundance of Firmicutes under treatments C2 and C3 was small. As the proportion of soft rock increased, the soil particles changed from point contact to surface contact, and the adhesion on the surface of the particles gradually increased. Results from this study show that the retention time of SOC can be increased and the carbon sequestration effect is better when the ratio of soft rock to sand is set to 1:2.

Proteomic analysis reveals the temperature-dependent presence of extracytoplasmic peptidases in the biofilm exoproteome of Listeria monocytogenes EGD-e: Yue-Jia Lee , Chinling Wang; J. Microbiol. 2020;58(9):761-771. Published online July 27, 2020; DOI: https://doi.org/10.1007/s12275-020-9522-8

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Abstract: The foodborne pathogen Listeria monocytogenes resists environmental stresses by forming biofilms. Because this pathogen transmits between the environment and the host, it must adapt to temperature as an environmental stress. In this study, we aimed to identify which proteins were present depending on the temperature in the biofilms of L. monocytogenes EGD-e. Proteins in the supernatants of biofilms formed at 25°C and 37°C were compared using two-dimensional gel electrophoresis and liquid chromatography with tandem mass spectrometry. The larger number of extracytoplasmic proteins associated with cell wall/membrane/envelop biogenesis was identified from the supernatant of biofilms formed at 25°C (7) than those at 37°C (0). Among the 16 extracytoplasmic proteins detected only at 25°C, three were peptidases, namely Spl, Cwh, and Lmo0186. Moreover, mRNA expression of the three peptidases was higher at 25°C than at 37°C. Interestingly, this adaptation of gene expression to temperature was present in sessile cells but not in dispersed cells. After inhibiting the activity of extracytoplasmic peptidases with a protease inhibitor, we noted that the levels of biofilm biomass increased with higher concentrations of the protease inhibitor only when L. monocytogenes grew biofilms at 25°C and not at 37°C. Overall, our data suggest an effect of temperature on the presence of peptidases in L. monocytogenes biofilms. Additionally, increasing the levels of extracytoplasmic peptidases in biofilms is likely a unique feature for sessile L. monocytogenes that causes a naturally occurring breakdown of biofilms and facilitates the pathogen exiting biofilms and disseminating into the environment.

Structural insights into the psychrophilic germinal protease PaGPR and its autoinhibitory loop: Chang Woo Lee , Saeyoung Lee , Chang-Sook Jeong , Jisub Hwang , Jeong Ho Chang , In-Geol Choi , T. Doohun Kim , HaJeung Park , Hye-Yeon Kim , Jun Hyuck Lee; J. Microbiol. 2020;58(9):772-779. Published online September 1, 2020; DOI: https://doi.org/10.1007/s12275-020-0292-0

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Abstract: In spore forming microbes, germination protease (GPR) plays a key role in the initiation of the germination process. A critical step during germination is the degradation of small acidsoluble proteins (SASPs), which protect spore DNA from external stresses (UV, heat, low temperature, etc.). Inactive zymogen GPR can be activated by autoprocessing of the N-terminal pro-sequence domain. Activated GPR initiates the degradation of SASPs; however, the detailed mechanisms underlying the activation, catalysis, regulation, and substrate recognition of GPR remain elusive. In this study, we determined the crystal structure of GPR from Paenisporosarcina sp. TG-20 (PaGPR) in its inactive form at a resolution of 2.5 Å. Structural analysis showed that the active site of PaGPR is sterically occluded by an inhibitory loop region (residues 202–216). The N-terminal region interacts directly with the self-inhibitory loop region, suggesting that the removal of the N-terminal pro-sequence induces conformational changes, which lead to the release of the self-inhibitory loop region from the active site. In addition, comparative sequence and structural analyses revealed that PaGPR contains two highly conserved Asp residues (D123 and D182) in the active site, similar to the putative aspartic acid protease GPR from Bacillus megaterium. The catalytic domain structure of PaGPR also shares similarities with the sequentially non-homologous proteins HycI and HybD. HycI and HybD are metalloproteases that also contain two Asp (or Glu) residues in their active site, playing a role in metal binding. In summary, our
results
provide useful insights into the activation process of PaGPR and its active conformation.

Chitosan-chelated zinc modulates cecal microbiota and attenuates inflammatory response in weaned rats challenged with Escherichia coli: Dan Feng , Minyang Zhang , Shiyi Tian , Jing Wang , Weiyun Zhu; J. Microbiol. 2020;58(9):780-792. Published online September 1, 2020; DOI: https://doi.org/10.1007/s12275-020-0056-x

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Abstract: Escherichia coli (E. coli) infection is very common among young growing animals, and zinc supplementation is often used to alleviate inflammation induced by this disease. Therefore, the objective of this study was to evaluate whether chitosan- chelated zinc (CS-Zn) supplementation could attenuate gut injury induced by E. coli challenge and to explore how CSZn modulates cecal microbiota and alleviates intestinal inflammation in weaned rats challenged with E. coli. 36 weaned rats (55.65 ± 2.18 g of BW, n = 12) were divided into three treatment groups consisting of unchallenged rats fed a basal diet (Control) and two groups of rats challenged with E. coli and fed a basal diet or a diet containing 640 mg/kg CS-Zn (E. coli + CS-Zn, containing 50 mg/kg Zn) for a 14-day experiment. On days 10 to 12, each rat was given 4 ml of E. coli solution with a total bacteria count of 1010 CFU by oral gavage daily or normal saline of equal dosage. CS-Zn supplementation mitigated intestinal morphology impairment (e.g. higher crypt depth and lower macroscopic damage index) induced by E. coli challenge (P < 0.05), and alleviated the increase of Myeloperoxidase (MPO) activity after E. coli challenge (P < 0.05). 16S rRNA sequencing analyses revealed that E. coli challenge significantly increased the abundance of Verrucomicrobia and E. coli (P < 0.05). However, CS-Zn supplementation increased the abundance of Lactobacillus and decreased the relative abundance of Proteobacteria, Desulfovibrio and E. coli (P < 0.05). The concentrations of butyrate in the cecal digesta, which decreased due to the challenge, were higher in the E. coli + CS-Zn group (P < 0.05). In addition, CS-Zn supplementation significantly prevented the elevation of pro-inflammatory cytokines IL-6 concentration and upregulated the level of anti-inflammatory cytokines IL-10 in cecal mucosa induced by E. coli infection (P < 0.05). In conclusion, these results indicate that CS-Zn produces beneficial effects in alleviating gut mucosal injury of E. coli challenged rats by enhancing the intestinal morphology and modulating cecal bacterial composition, as well as attenuating inflammatory response.

Omp16, a conserved peptidoglycan-associated lipoprotein, is involved in Brucella virulence in vitro: Feijie Zhi , Dong Zhou , Junmei Li , Lulu Tian , Guangdong Zhang , Yaping Jin , Aihua Wang; J. Microbiol. 2020;58(9):793-804. Published online September 1, 2020; DOI: https://doi.org/10.1007/s12275-020-0144-y

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Abstract: Brucella, the bacterial agent of common zoonotic brucellosis, primarily infects specific animal species. The Brucella outer membrane proteins (Omps) are particularly attractive for developing vaccine and improving diagnostic tests and are associated with the virulence of smooth Brucella strains. Omp16 is a homologue to peptidoglycan-associated lipoproteins (Pals), and an omp16 mutant has not been generated in any Brucella strain until now. Very little is known about the functions and pathogenic mechanisms of Omp16 in Brucella. Here, we confirmed that Omp16 has a conserved Pal domain and is highly conserved in Brucella. We attempted to delete omp16 in Brucella suis vaccine strain 2 (B. suis S2) without success, which shows that Omp16 is vital for Brucella survival. We acquired a B. suis S2 Omp16 mutant via conditional complementation. Omp16 deficiency impaired Brucella outer membrane integrity and activity in vitro. Moreover, inactivation of Omp16 decreased bacterial intracellular survival in macrophage RAW 264.7 cells. B. suis S2 and its derivatives induced marked expression of IL-1β, IL-6, and TNF-α mRNA in Raw 264.7 cells. Whereas inactivation of Omp16 in Brucella enhanced IL-1β and IL-6 expression in Raw 264.7 cells. Altogether, these findings show that the Brucella Omp16 mutant was obtained via conditional complementation and confirmed that Omp16 can maintain outer membrane integrity and be involved in bacterial virulence in Brucella in vitro and in vivo. These results will be important in uncovering the pathogenic mechanisms of Brucella.

Evolutionary analysis and protein family classification of chitin deacetylases in Cryptococcus neoformans: Seungsue Lee , Hyun Ah Kang , Seong-il Eyun; J. Microbiol. 2020;58(9):805-811. Published online September 1, 2020; DOI: https://doi.org/10.1007/s12275-020-0288-9

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Abstract: Cryptococcus neoformans is an opportunistic fungal pathogen causing cryptococcal meningoencephalitis. Interestingly, the cell wall of C. neoformans contains chitosan, which is critical for its virulence and persistence in the mammalian host. C. neoformans (H99) has three chitin deacetylases (CDAs), which convert chitin to chitosan. Herein, the classification of the chitin-related protein (CRP) family focused on cryptococcal CDAs was analyzed by phylogenetics, evolutionary pressure (dN/dS), and 3D modeling. A phylogenetic tree of 110 CRPs revealed that they can be divided into two clades, CRP I and II with bootstrap values (> 99%). CRP I clade comprises five groups (Groups 1–5) with a total of 20 genes, while CRP II clade comprises sixteen groups (Groups 6–21) with a total of 90 genes. CRP I comprises only fungal CDAs, including all three C. neoformans CDAs, whereas CRP II comprises diverse CDAs from fungi, bacteria, and amoeba, along with other carbohydrate esterase 4 family proteins. All CDAs have the signal peptide, except those from group 11. Notably, CDAs with the putative O-glycosylation site possess either the glycosylphosphatidylinositol (GPI)-anchor motif for CRP I or the chitin-binding domain (CBD) for CRP II, respectively. This evolutionary conservation strongly indicates that the O-glycosylation modification and the presence of either the GPI-anchor motif or the chitin-binding domain is important for fungal CDAs to function efficiently at the cell surface. This study reveals that C. neoformans CDAs carrying GPI anchors have evolved divergently from fungal and bacterial CDAs, providing new insights into evolution and classification of CRP family.

Caspase-3 inhibitor inhibits enterovirus D68 production: Wenbo Huo , Jinghua Yu , Chunyu Liu , Ting Wu , Yue Wang , Xiangling Meng , Fengmei Song , Shuxia Zhang , Ying Su , Yumeng Liu , Jinming Liu , Xiaoyan Yu , Shucheng Hua; J. Microbiol. 2020;58(9):812-820. Published online September 1, 2020; DOI: https://doi.org/10.1007/s12275-020-0241-y

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Abstract: Enterovirus D68 (EVD68) is an emerging pathogen that recently caused a large worldwide outbreak of severe respiratory disease in children. However, the relationship between EVD68 and host cells remains unclear. Caspases are involved in cell death, immune response, and even viral production. We found that caspase-3 was activated during EVD68 replication to induce apoptosis. Caspase-3 inhibitor (Z-DEVDFMK) inhibited viral production, protected host cells from the cytopathic effects of EVD68 infection, and prevented EVD68 from regulating the host cell cycle at G0/G1. Meanwhile, caspase-3 activator (PAC-1) increased EVD68 production. EVD68 infection therefore activates caspase-3 for virus production. This knowledge provides a potential direction for the prevention and treatment of disease related to EVD68.

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