Journal of Microbiology

Review

Bacterial Sialic Acid Catabolism at the Host–Microbe Interface: Jaeeun Kim , Byoung Sik Kim; J. Microbiol. 2023;61(4):369-377. Published online March 27, 2023; DOI: https://doi.org/10.1007/s12275-023-00035-7

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Sialic acids consist of nine-carbon keto sugars that are commonly found at the terminal end of mucins. This positional feature of sialic acids contributes to host cell interactions but is also exploited by some pathogenic bacteria in evasion of host immune system. Moreover, many commensals and pathogens use sialic acids as an alternative energy source to survive within the mucus-covered host environments, such as the intestine, vagina, and oral cavity. Among the various biological events mediated by sialic acids, this review will focus on the processes necessary for the catabolic utilization of sialic acid in bacteria. First of all, transportation of sialic acid should be preceded before its catabolism. There are four types of transporters that are used for sialic acid uptake; the major facilitator superfamily (MFS), the tripartite ATP-independent periplasmic C4-dicarboxilate (TRAP) multicomponent transport system, the ATP binding cassette (ABC) transporter, and the sodium solute symporter (SSS). After being moved by these transporters, sialic acid is degraded into an intermediate of glycolysis through the well-conserved catabolic pathway. The genes encoding the catabolic enzymes and transporters are clustered into an operon(s), and their expression is tightly controlled by specific transcriptional regulators. In addition to these mechanisms, we will cover some researches about sialic acid utilization by oral pathogens.

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Journal Articles

Relaxed Cleavage Specificity of Hyperactive Variants of Escherichia coli RNase E on RNA I: Dayeong Bae , Hana Hyeon , Eunkyoung Shin , Ji&# , Kangseok Lee; J. Microbiol. 2023;61(2):211-220. Published online February 22, 2023; DOI: https://doi.org/10.1007/s12275-023-00013-z

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Abstract

RNase E is an essential enzyme in Escherichia coli. The cleavage site of this single-stranded specific endoribonuclease is well-characterized in many RNA substrates. Here, we report that the upregulation of RNase E cleavage activity by a mutation that affects either RNA binding (Q36R) or enzyme multimerization (E429G) was accompanied by relaxed cleavage specificity. Both mutations led to enhanced RNase E cleavage in RNA I, an antisense RNA of ColE1-type plasmid replication, at a major site and other cryptic sites. Expression of a truncated RNA I with a major RNase E cleavage site deletion at the 5′-end (RNA I- 5) resulted in an approximately twofold increase in the steady-state levels of RNA I- 5 and the copy number of ColE1-type plasmid in E. coli cells expressing wild-type or variant RNase E compared to those expressing RNA I. These
results
indicate that RNA I- 5 does not efficiently function as an antisense RNA despite having a triphosphate group at the 5′-end, which protects the RNA from ribonuclease attack. Our study suggests that increased cleavage rates of RNase E lead to relaxed cleavage specificity on RNA I and the inability of the cleavage product of RNA I as an antisense regulator in vivo does not stem from its instability by having 5′-monophosphorylated end.

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Engineering an Escherichia coli based in vivo mRNA manufacturing platform
Edward Curry, George Muir, Jixin Qu, Zoltán Kis, Martyn Hulley, Adam Brown
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Characterization of a novel phage depolymerase specific to Escherichia coli O157:H7 and biofilm control on abiotic surfaces: Do-Won Park , Jong-Hyun Park; J. Microbiol. 2021;59(11):1002-1009. Published online October 6, 2021; DOI: https://doi.org/10.1007/s12275-021-1413-0

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Abstract

The increasing prevalence of foodborne diseases caused by Escherichia coli O157:H7 as well as its ability to form biofilms poses major threats to public health worldwide. With increasing concerns about the limitations of current disinfectant treatments, phage-derived depolymerases may be used as promising biocontrol agents. Therefore, in this study, the characterization, purification, and application of a novel phage depolymerase, Dpo10, specifically targeting the lipopolysaccharides of E. coli O157, was performed. Dpo10, with a molecular mass of 98 kDa, was predicted to possess pectate lyase activity via genome analysis and considered to act as a receptor- binding protein of the phage. We confirmed that the purified Dpo10 showed O-polysaccharide degrading activity only for the E. coli O157 strains by observing its opaque halo. Dpo10 maintained stable enzymatic activities across a wide range of temperature conditions under 55°C and mild basic pH. Notably, Dpo10 did not inhibit bacterial growth but significantly increased the complement-mediated serum lysis of E. coli O157 by degrading its O-polysaccharides. Moreover, Dpo10 inhibited the biofilm formation against E. coli O157 on abiotic polystyrene by 8-fold and stainless steel by 2.56 log CFU/coupon. This inhibition was visually confirmed via fieldemission scanning electron microscopy. Therefore, the novel depolymerase from E. coli siphophage exhibits specific binding and lytic activities on the lipopolysaccharide of E. coli O157 and may be used as a promising anti-biofilm agent against the E. coli O157:H7 strain.

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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.

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Review

[Minireview]Recent advances in genetic engineering tools based on synthetic biology: Jun Ren , Jingyu Lee , Dokyun Na; J. Microbiol. 2020;58(1):1-10. Published online January 2, 2020; DOI: https://doi.org/10.1007/s12275-020-9334-x

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Abstract

Genome-scale engineering is a crucial methodology to rationally regulate microbiological system operations, leading to expected biological behaviors or enhanced bioproduct yields. Over the past decade, innovative genome modification technologies have been developed for effectively regulating and manipulating genes at the genome level. Here, we discuss the current genome-scale engineering technologies used for microbial engineering. Recently developed strategies, such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, multiplex automated genome engineering (MAGE), promoter engineering, CRISPR-based regulations, and synthetic small regulatory RNA (sRNA)-based knockdown, are considered as powerful tools for genome-scale engineering in microbiological systems. MAGE, which modifies specific nucleotides of the genome sequence, is utilized as a genome-editing tool. Contrastingly, synthetic sRNA, CRISPRi, and CRISPRa are mainly used to regulate gene expression without modifying the genome sequence. This review introduces the recent genome-scale editing and regulating technologies and their applications in metabolic engineering.

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Journal Articles

Identification and characterization of a novel light-induced promoter for recombinant protein production in Pleurotus ostreatus: Chaomin Yin , Xiuzhi Fan , Kun Ma , Zheya Chen , Defang Shi , Fen Yao , Hong Gao , Aimin Ma; J. Microbiol. 2020;58(1):39-45. Published online November 4, 2019; DOI: https://doi.org/10.1007/s12275-020-9230-4

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Abstract

A lectin gene (plectin) with a high level of expression was previously identified by comparative transcriptome analysis of Pleurotus ostreatus. In this study, we cloned a 733-bp DNA fragment from the start codon of the plectin gene. Sequence analysis showed that the plectin promoter (Plp) region contained several eukaryotic transcription factor binding motifs, such as the TATA-box, four possible CAAT-box, light responsiveness motifs and MeJA-responsiveness motifs. To determine whether the Plp promoter was a light-regulated promoter, we constructed an expression vector with the fused egfp-hph fragment under the control of the Plp promoter and transformed P. ostreatus mycelia via Agrobacterium tumefaciens. PCR and Southern blot analyses confirmed the Plpegfp- hph fragment was integrated into the chromosomal DNA of transformants. qRT-PCR, egfp visualization, and intracellular egfp determination experiments showed the Plp promoter could be a light-induced promoter that may be suitable for P. ostreatus genetic engineering. This study lays the foundation for gene homologous expression in P. ostreatus.

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The GATA transcription factor BcWCL2 regulates citric acid secretion to maintain redox homeostasis and full virulence in Botrytis cinerea
Weiheng Ren, Chen Qian, Dandan Ren, Yunfei Cai, Zhaohui Deng, Ning Zhang, Congcong Wang, Yiwen Wang, Pinkuan Zhu, Ling Xu, Regine Kahmann
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Evaluation and application of constitutive promoters for cutinase production by Saccharomyces cerevisiae: Juan Zhang , Yanqiu Cai , Guocheng Du , Jian Chen , Miao Wang , Zhen Kang; J. Microbiol. 2017;55(7):538-544. Published online June 30, 2017; DOI: https://doi.org/10.1007/s12275-017-6514-4

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Abstract

died and applied in processes targeted for industrial scale. In this work, the cutinase gene tfu from Thermobifida fusca was artificially synthesized according to codon usage bias of Saccharomyces cerevisiae and investigated in Saccharomyces cerevisiae. Using the α-factor signal peptide, the T. fusca cutinase was successfully overexpressed and secreted with the GAL1 expression system. To increase the cutinase level and overcome some of the drawbacks of induction, four different strong promoters (ADH1, HXT1, TEF1, and TDH3) were comparatively evaluated for cutinase production. By comparison, promoter TEF1 exhibited an outstanding property and significantly increased the expression level. By fed-batch fermentation with a constant feeding approach, the activity of cutinase was increased to 29.7 U/ml. The result will contribute to apply constitutive promoter TEF1 as a tool for targeted cutinase production in S. cerevisiae cell factory.

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

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.

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Sequence-dependent model of genes with dual σ factor preference
Ines S.C. Baptista, Vinodh Kandavalli, Vatsala Chauhan, Mohamed N.M. Bahrudeen, Bilena L.B. Almeida, Cristina S.D. Palma, Suchintak Dash, Andre S. Ribeiro
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms.2022; 1865(3): 194812. CrossRef
Function Enhancement of a Metabolic Module via Endogenous Promoter Replacement for Pseudomonas sp. JY-Q to Degrade Nicotine in Tobacco Waste Treatment
Jun Li, Fengmei Yi, Guoqing Chen, Fanda Pan, Yang Yang, Ming Shu, Zeyu Chen, Zeling Zhang, Xiaotong Mei, Weihong Zhong
Applied Biochemistry and Biotechnology.2021; 193(9): 2793. CrossRef
Recent advances in genetic engineering tools based on synthetic biology
Jun Ren, Jingyu Lee, Dokyun Na
Journal of Microbiology.2020; 58(1): 1. CrossRef

Hypermethylation of the interferon regulatory factor 5 promoter in Epstein-Barr virus-associated gastric carcinoma: Seung Myung Dong , Hyun Gyu Lee , Sung-Gyu Cho , Seung-Hyun Kwon , Heejei Yoon , Hyun-Jin Kwon , Ji Hae Lee , Hyemi Kim , Pil-Gu Park , Hoguen Kim , S. Diane Hayward , Jeon Han Park , Jae Myun Lee; J. Microbiol. 2015;53(1):70-76. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-014-4654-3

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Abstract

Interferon regulatory factor-5 (IRF-5), a member of the mammalian IRF transcription factor family, is regulated by p53, type I interferon and virus infection. IRF-5 participates in virus-induced TLR-mediated innate immune responses and may play a role as a tumor suppressor. It was suppressed in various EBV-infected transformed cells, thus it is valuable to identify the suppression mechanism. We focused on a promoter CpG islands methylation, a kind of epigenetic regulation in EBV-associated Burkitt’s lymphomas (BLs) and gastric carcinomas. IRF-5 is not detected in most of EBV-infected BL cell lines due to hypermethylation of IRF-5 distal promoter (promoter-A), which was restored by a demethylating agent, 5-aza-2􍿁-deoxycytidine. Hypomethylation of CpG islands in promoter-A was observed only in EBV type III latent infected BL cell lines (LCL and Mutu III). Similarly, during EBV infection to Akata-4E3 cells, IRF-5 was observed at early time periods (2 days to 8 weeks), concomitant unmethylation of promoter-A, but suppressed in later infection periods as observed in latency I BL cell lines. Moreover, hypermethylation in IRF-5 promoter-A region was also observed in EBV-associated gastric carcinoma (EBVaGC) cell lines or primary gastric carcinoma tissues, which show type I latent infection. In summary, IRF-5 is suppressed by hypermethylation of its promoter-A in most of EBV-infected transformed cells, especially BLs and EBVaGC. EBV-induced carcinogenesis takes an advantage of proliferative effects of TLR signaling, while limiting IRF-5 mediated negative effects in the establishment of EBVaGCs.

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A Potent Brucella abortus 2308 Δery Live Vaccine Allows for the Differentiation between Natural and Vaccinated Infection: Junbo Zhang , Shuanghong Yin , Fei Guo , Ren Meng , Chuangfu Chen , Hui Zhang , Zhiqiang Li , Qiang Fu , Huijun Shi , Shengwei Hu , Wei Ni , Tiansen Li , Ke Zhang; J. Microbiol. 2014;52(8):681-688. Published online July 4, 2014; DOI: https://doi.org/10.1007/s12275-014-3689-9

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Abstract

Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. However, the current Brucella abortus vaccines (S19 and RB51) are deficient; they can cause abortion in pregnant animals. Moreover, when the vaccine S19 is used, tests cannot differentiate natural from vaccinated infection. Therefore, a safer and more potent vaccine is needed. A Brucella abortus 2308 ery promoter mutant (Δery) was constructed to overcome these drawbacks. The growth of the Δery mutant was significantly attenuated in macrophages and mice and induced high protective immunity in mice. Moreover, Δery induced an anti-Brucellaspecific IgG (immunoglobulin G) response and stimulated the expression of interferon-gamma (INF-γ) and interleukin-4 (IL-4). Furthermore, the expression of EryA antigen allowed for the serological differentiation between natural and vaccinated infection in mice. These results indicate that the Δery mutant is a potential attenuated live vaccine candidate against virulent Brucella abortus 2308 (S2308) infection.

Citations

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Novel Bifidobacterium Promoters Selected Through Microarray Analysis Lead to Constitutive High-Level Gene Expression: Yan Wang , Jin Yong Kim , Myeong Soo Park , Geun Eog Ji; J. Microbiol. 2012;50(4):638-643. Published online July 21, 2012; DOI: https://doi.org/10.1007/s12275-012-1591-x

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Abstract: For the development of a food-grade expression system for Bifidobacterium, a strong promoter leading to high-level expression of cloned gene is a prerequisite. For this purpose, a promoter screening host-vector system for Bifidobacterium has been established using β-glucosidase from Bifidobacterium lactis as a reporter and Bifidobacterium bifidum BGN4 as a host, which is β-glucosidase negative strain. Seven putative promoters showing constitutive high-level expression were selected through microarray analysis based on the genome sequence of B. bifidum BGN4. They were cloned into upstream of β-glucosidase gene and transformed into Escherichia coli DH5α and B. bifidum BGN4. Promoter activities were analyzed both in E. coli and B. bifidum BGN4 by measuring β-glucosidase activity. β-Glucosidase activities in all of the transformants showed growth-associated characteristics. Among them, P919 was the strongest in B. bifidum BGN4 and showed maximum activity at 18 h, while P895 was the strongest in E. coli DH5α at 7 h. This study shows that novel strong promoters such as P919 can be used for high-level expression of foreign genes in Bifidobacterium and will be useful for the construction of an efficient food-grade expression system.

Journal Article

Characterization of Trichoderma reesei Endoglucanase II Expressed Heterologously in Pichia pastoris for Better Biofinishing and Biostoning: Sutanu Samanta , Asitava Basu , Umesh Chandra Halder , Soumitra Kumar Sen; J. Microbiol. 2012;50(3):518-525. Published online June 30, 2012; DOI: https://doi.org/10.1007/s12275-012-1207-5

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Abstract: The endoglucanase II of Trichoderma reesei is considered the most effective enzyme for biofinishing cotton fabrics and biostoning denim garments. However, the commercially available preparation of endoglucanase II is usually mixed with other cellulase components, especially endoglucanase I, resulting in hydrolysis and weight loss of garments during biofinishing and biostoning. We thus isolated the endoglucanase II gene from T. reesei to express this in Pichia pastoris, under the control of a methanol-inducible AOX1 promoter, to avoid the presence of other cellulase components. A highly expressible Mut+ transformant was selected and its expression in BMMH medium was found most suitable for the production of large amounts of the recombinant protein. Recombinant endoglucanase II was purified to electrophoretic homogeneity, and functionally characterized by activity staining. The specific activity of recombinant endoglucanase II was found to be 220.57 EU/mg of protein. Purified recombinant endoglucanase II was estimated to have a molecular mass of 52.8 kDa. The increase in molecular mass was likely due to hyperglycosylation. Hyperglycosylation of recombinant endoglucanase II secreted by P. pastoris did not change the temperature or pH optima as compared to the native protein, but did result in increased thermostability. Kinetic analysis showed that recombinant endoglucanase was most active against amorphous cellulose, such as carboxymethyl cellulose, for which it also had a high affinity.

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

Promoter Analysis of Bombyx mori Nucleopolyhedrovirus Ubiquitin Gene: Xu’ai Lin , Yin Chen , Yongzhu Yi , Jie Yan , Zhifang Zhang; J. Microbiol. 2008;46(4):429-435. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-007-0163-y

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Abstract: The aim of this study was to analyze the characteristics of Bombyx mori nucleopolyhedrovirus (BmNPV) ubiquitin gene promoter and the effects of conserved motifs, such as TAAG, TATA, and CAAT, along with baculovirus enhancer homologous region 3 (hr3), on promoter activity. Ubiquitin gene of BmNPV was expressed during the late phase of virus infection. In the presence of viral factors, significant reduction of promoter activity was observed by deletion of -382 to -124 bp upstream of ATG. The fragment between -187 and -383 bp upstream of ATG, including distal TAAG, CAAT motif, and TATA box, could also drive expression of the reporter gene. The mutation of cis-elements TATA boxes and TAAG motifs significantly decreased the promoter’s activity, while CAAT mutations enhanced promoter activity by 2- or 3-fold, as compared with the native promoter. In the presence of BmNPV, hr3, both located downstream of the reporter gene of the same vector, and separate vector, could significantly enhance transcription activity of ubiquitin promoter as compared to the control. We concluded that BmNPV ubiquitin gene might be regulated by dual sets of promoter elements, where TAAG and TATA box may positively regulate the expression of ubiquitin, while CAAT motif functions as a negative regulator. Viral factor(s) play an important role in the co-activation of hr3 and promoter.

Cloning and Sequence Analysis of a Glyceraldehyde-3-phosphate Dehydrogenase Gene from Ganoderma lucidum: Xu Fei , Ming Wen Zhao , Yu Xiang Li; J. Microbiol. 2006;44(5):515-522.; DOI: https://doi.org/2446 [pii]

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Abstract: A cDNA library of Ganoderma lucidum has been constructed using a Zap Express cloning vector. A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR. By comparison of the cDNA and the genomic DNA sequences, it was found that the complete nucleotide sequence encodes a putative polypeptide chain of 338 amino acids interrupted by 6 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from yeast and filamentous fungi. The promoter region contains a CT-rich stretch, two CAAT boxes, and a consensus TATA box. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.

Journal Article

Computational Detection of Prokaryotic Core Promoters in Genomic Sequences: Ki-Bong Kim , Jeong Seop Sim; J. Microbiol. 2005;43(5):411-416.; DOI: https://doi.org/2282 [pii]

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Abstract: The high-throughput sequencing of microbial genomes has resulted in the relatively rapid accumulation of an enormous amount of genomic sequence data. In this context, the problem posed by the detection of promoters in genomic DNA sequences via computational methods has attracted considerable research attention in recent years. This paper addresses the development of a predictive model, known as the dependence decomposition weight matrix model (DDWMM), which was designed to detect the core promoter region, including the -10 region and the transcription start sites (TSSs), in prokaryotic genomic DNA sequences. This is an issue of some importance with regard to genome annotation efforts. Our predictive model captures the most significant dependencies between positions (allowing for non-adjacent as well as adjacent dependencies) via the maximal dependence decomposition (MDD) procedure, which iteratively decomposes data sets into subsets, based on the significant dependence between positions in the promoter region to be modeled. Such dependencies may be intimately related to biological and structural concerns, since promoter elements are present in a variety of combinations, which are separated by various distances. In this respect, the DDWMM may prove to be appropriate with regard to the detection of core promoter regions and TSSs in long microbial genomic contigs. In order to demonstrate the effectiveness of our predictive model, we applied 10-fold cross-validation experiments on the 607 experimentally-verified promoter sequences, which evidenced good performance in terms of sensitivity.

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