Journal of Microbiology

Journal Articles

Development of a Novel D‑Lactic Acid Production Platform Based on Lactobacillus saerimneri TBRC 5746: Kitisak Sansatchanon , Pipat Sudying , Peerada Promdonkoy , Yutthana Kingcha , Wonnop Visessanguan , Sutipa Tanapongpipat , Weerawat Runguphan , Kanokarn Kocharin; J. Microbiol. 2023;61(9):853-863. Published online September 14, 2023; DOI: https://doi.org/10.1007/s12275-023-00077-x

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Abstract: D-Lactic acid is a chiral, three-carbon organic acid, that bolsters the thermostability of polylactic acid. In this study, we developed a microbial production platform for the high-titer production of D-lactic acid. We screened 600 isolates of lactic acid bacteria (LAB) and identified twelve strains that exclusively produced D-lactic acid in high titers. Of these strains, Lactobacillus saerimneri TBRC 5746 was selected for further development because of its homofermentative metabolism. We investigated the effects of high temperature and the use of cheap, renewable carbon sources on lactic acid production and observed a titer of 99.4 g/L and a yield of 0.90 g/g glucose (90% of the theoretical yield). However, we also observed L-lactic acid production, which reduced the product’s optical purity. We then used CRISPR/dCas9-assisted transcriptional repression to repress the two Lldh genes in the genome of L. saerimneri TBRC 5746, resulting in a 38% increase in D-lactic acid production and an improvement in optical purity. This is the first demonstration of CRISPR/dCas9-assisted transcriptional repression in this microbial host and represents progress toward efficient microbial production of D-lactic acid.

Metformin Regulates Gut Microbiota Abundance to Suppress M2 Skewing of Macrophages and Colorectal Tumorigenesis in Mice: Linfeng Fan , Xiangfu Zeng , Guofeng Xu; J. Microbiol. 2023;61(1):109-120. Published online January 26, 2023; DOI: https://doi.org/10.1007/s12275-022-00010-8

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Abstract: The correlation of imbalanced gut microbiota with the onset and progression of colorectal cancer (CRC) has become clear. This work investigates the effect of metformin on gut microbiota and genesis of CRC in mice. Human fecal samples were collected from healthy control (HC) donors and CRC patients. Compared to HC donors, CRC patients had reduced abundance of gut microbiota; however, they had increased abundance of detrimental Bacteroidetes. Mice were injected with azomethane (AOM) to induce colorectal tumorigenesis models. Treatment of CRC patients-sourced fecal microbiota promoted tumorigenesis, and it increased the expression of Ki67, β-catenin, COX-2, and Cyclin D1 in mouse colon tissues. Further treatment of metformin blocked the colorectal tumorigenesis in mice. Fecal microbiota from the metformin-treated mice was collected, which showed decreased Bacteroidetes abundance and suppressed AOM-induced colorectal tumorigenesis in mice as well. Moreover, the metformin- modified microbiota promoted the M1 macrophage-related markers IL-6 and iNOS but suppressed the M2 macrophage-related markers IL-4R and Arg1 in mouse colon tissues. In conclusion, this study suggests that metformin-mediated gut microbiota alteration suppresses macrophage M2 polarization to block colorectal tumorigenesis.

[PROTOCOL]Analyzing viral epitranscriptomes using nanopore direct RNA sequencing: Ari Hong , Dongwan Kim , V. Narry Kim , Hyeshik Chang; J. Microbiol. 2022;60(9):867-876. Published online August 24, 2022; DOI: https://doi.org/10.1007/s12275-022-2324-4

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Abstract: RNA modifications are a common occurrence across all domains of life. Several chemical modifications, including N6- methyladenosine, have also been found in viral transcripts and viral RNA genomes. Some of the modifications increase the viral replication efficiency while also helping the virus to evade the host immune system. Nonetheless, there are numerous examples in which the host's RNA modification enzymes function as antiviral factors. Although established methods like MeRIP-seq and miCLIP can provide a transcriptome- wide overview of how viral RNA is modified, it is difficult to distinguish between the complex overlapping viral transcript isoforms using the short read-based techniques. Nanopore direct RNA sequencing (DRS) provides both long reads and direct signal readings, which may carry information about the modifications. Here, we describe a refined protocol for analyzing the RNA modifications in viral transcriptomes using nanopore technology.

Analysis of a bac operon-silenced strain suggests pleiotropic effects of bacilysin in Bacillus subtilis: Ozan Ertekin , Meltem Kutnu , Aslı Aras Ta&# , Mustafa Demir , Ayten Yazgan Karata&# , Gülay Özcengiz; J. Microbiol. 2020;58(4):297-313. Published online January 28, 2020; DOI: https://doi.org/10.1007/s12275-020-9064-0

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Abstract: Bacilysin, as the simplest peptide antibiotic made up of only L-alanine and L-anticapsin, is produced and excreted by Bacillus subtilis under the control of quorum sensing. We analyzed bacilysin-nonproducing strain OGU1 which was obtained by bacA-targeted pMutin T3 insertion into the parental strain genome resulting in a genomic organization (bacA􍿁::lacZ::erm::bacABCDEF) to form an IPTG-inducible bac operon. Although IPTG induction provided 3- to 5-fold increment in the transcription of bac operon genes, no bacilysin activity was detectable in bioassays and inability of the OGU1 to form bacilysin was confirmed by UPLC-mass spectrometry analysis. Phenotypic analyses revealed the deficiencies in OGU1 with respect to colony pigmentation, spore coat proteins, spore resistance and germination, which could be rescued by external addition of bacilysin concentrate into its cultures. 2DE MALDI-TOF/MS and nanoLC-MS/MS were used as complementary approaches to compare cytosolic proteomes of OGU1. 2-DE identified 159 differentially expressed proteins corresponding to 121 distinct ORFs. In nanoLCMS/ MS, 76 proteins were differentially expressed in OGU1. Quantitative transcript analyses of selected genes validated the proteomic findings. Overall, the results pointed to the impact of bacilysin on expression of certain proteins of sporulation and morphogenesis; the members of mother cell compartment- specific σE and σK regulons in particular, quorum sensing and two component-global regulatory systems, peptide transport, stress response as well as CodY- and ScoCregulated proteins.

Transcriptomic and proteomic profiling revealed global changes in Streptococcus thermophilus during pH-controlled batch fermentations: Yali Qiao , Cong Leng , Gefei Liu , Yanjiao Zhang , Xuepeng Lv , Hongyu Chen , Jiahui Sun , Zhen Feng; J. Microbiol. 2019;57(9):769-780. Published online June 14, 2019; DOI: https://doi.org/10.1007/s12275-019-8604-y

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Abstract: Understanding global changes of physiological processes at the molecular level during the growth of Streptococcus thermophilus is essential for the rational design of cultivation media and the optimization of bioprocesses. Transcriptomics and proteomics were combined to investigate the global changes at the transcript and protein level during the growth of S. thermophilus. The expression of 1396 genes (FDR ≤ 0.001) and 876 proteins (P < 0.05) changed significantly over time. The most remarkable growth phase dependent changes occurred in the late-lag phase and were related to heterofermentation, glycolysis, peptidoglycan biosynthesis, conversion between amino acids and stress response. The present
results
could provide theoretical guidance for high-cell-density culture, help design cultivation media, and help attain a high biomass of S. thermophilus.

Review

MINIREVIEW] Cure of tuberculosis using nanotechnology: An overview: Rout George Kerry , Sushanto Gouda , Bikram Sil , Gitishree Das , Han-Seung Shin , Gajanan Ghodake , Jayanta Kumar Patra; J. Microbiol. 2018;56(5):287-299. Published online May 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7414-y

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Abstract: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a major health issue of the present era. The bacterium inhabits the host macrophage and other immune cells where it modulates the lysosome trafficking protein, hinders the formation of phagolysosome, and blocks the TNF receptor- dependent apoptosis of host macrophage/monocytes. Other limitations such as resistance to and low bioavailability and bio-distribution of conventional drugs aid to their high virulence and human mortality. This review highlights the use of nanotechnology-based approaches for drug formulation and delivery which could open new avenues to limit the pathogenicity of tuberculosis. Moreover phytochemicals, such as alkaloids, phenols, saponins, steroids, tannins, and terpenoids, extracted from terrestrial plants and mangroves seem promising against M. tuberculosis through different molecular mechanisms. Further understanding of the genomics and proteomics of this pathogenic microbe could also help overcome various research gaps in the path of developing a suitable therapy against tuberculosis.

Journal Articles

Proteome analysis reveals global response to deletion of mrflbA in Monascus ruber: Qingqing Yan , Zhouwei Zhang , Yishan Yang , Fusheng Chen , Yanchun Shao; J. Microbiol. 2018;56(4):255-263. Published online February 28, 2018; DOI: https://doi.org/10.1007/s12275-018-7425-8

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Abstract: Monascus spp. are commonly used for a wide variety of applications in the food and pharmaceutical industries. In previous studies, the knock-out of mrflbA (a putative regulator of the G protein α subunit) in M. ruber led to autolysis of the mycelia, decreased pigmentation and lowered mycotoxin production. Therefore, we aimed to obtain a comprehensive overview of the underlying mechanism of mrflbA deletion at the proteome level. A two-dimensional gel electrophoresis analysis of mycelial proteins indicated that the abundance of 178 proteins was altered in the ΔmrflbA strain, 33 of which were identified with high confidence. The identified proteins are involved in a range of activities, including carbohydrate and amino acid metabolism, hyphal development and the oxidative stress response, protein modification, and the regulation of cell signaling. Consistent with these findings, the activity of antioxidative enzymes and chitinase was elevated in the supernatant of the ΔmrflbA strain. Furthermore, deletion of mrflbA resulted in the transcriptional reduction of secondary metabolites (pigment and mycotoxin). In short, the mutant phenotypes induced by the deletion of mrflbA were consistent with changes in the expression levels of associated proteins, providing direct evidence of the regulatory functions mediated by mrflbA in M. ruber.

The protein and neutral lipid composition of lipid droplets isolated from the fission yeast, Schizosaccharomyces pombe: Alex Meyers , Karuna Chourey , Taylor M. Weiskittel , Susan Pfiffner , John R. Dunlap , Robert L. Hettich , Paul Dalhaimer; J. Microbiol. 2017;55(2):112-122. Published online January 26, 2017; DOI: https://doi.org/10.1007/s12275-017-6205-1

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Abstract: Lipid droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer with bound proteins. Much of the information on lipid droplet function comes from proteomic and lipodomic studies that identify the components of droplets isolated from organisms throughout the phylogenetic tree. Here, we add to that important inventory by reporting lipid droplet factors from the fission yeast, Schizosaccharomyces pombe. Unique to this study was the fact that cells were cultured in three different environments: 1) late log growth phase in glucose-based media, 2) stationary phase in glucosebased media, and 3) late log growth phase in media containing oleic acid. We confirmed colocalization of major factors with lipid droplets using live-cell fluorescent microscopy. We also analyzed droplets from each of the three conditions for sterol ester (SE) and triacylglycerol (TAG) content, along with their respective fatty acid compositions. We identified a previously undiscovered lipid droplet protein, Vip1p, which affects droplet size distribution. The results provide further insight into the workings of these ubiquitous organelles.

Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium Novosphingobium pentaromativorans US6-1: Sung Ho Yun , Sang-Yeop Lee , Chi-Won Choi , Hayoung Lee , Hyun-Joo Ro , Sangmi Jun , Yong Min Kwon , Kae Kyoung Kwon , Sang-Jin Kim , Gun-Hwa Kim , Seung Il Kim; J. Microbiol. 2017;55(1):56-62. Published online December 30, 2016; DOI: https://doi.org/10.1007/s12275-017-6581-6

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18 Citations

Abstract: Novosphingobium pentaromativorans US6-1 is a Gram-negative halophilic marine bacterium able to utilize several polycyclic aromatic hydrocarbons such as phenanthrene, pyrene, and benzo[a]pyrene. In this study, using transmission electron microscopy, we confirmed that N. pentaromativorans US6-1 produces outer membrane vesicles (OMVs). N. pentaromativorans OMVs (hereafter OMVNovo) are spherical in shape, and the average diameter of OMVNovo is 25–70 nm. Proteomic analysis revealed that outer membrane proteins and periplasmic proteins of N. pentaromativorans are the major protein components of OMVNovo. Comparative proteomic analysis with the membrane-associated protein fraction and correlation analysis demonstrated that the outer membrane proteins of OMVNovo originated from the membrane- associated protein fraction. To the best of our knowledge, this study is the first to characterize OMV purified from halophilic marine bacteria.

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

Proteomic and Functional Analyses of a Novel Porin-like Protein in Xanthomonas oryzae pv. oryzae: Hye-Jee Park , Sang-Won Lee , Sang-Wook Han; J. Microbiol. 2014;52(12):1030-1035. Published online November 29, 2014; DOI: https://doi.org/10.1007/s12275-014-4442-0

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Abstract: Proteomic analysis is a useful technique for postulating and elucidating protein functions. In the present work, a shotgun proteomic analysis was used to identify functions of the PXO_03968 gene (previously known as the ax21) from Xanthomonas oryzae pv. oryzae (Xoo), a causal agent for bacterial blight disease in rice. Structural prediction performed on the protein sequence encoded by PXO_03968 reveals that it encodes a putative porin-like protein, possessing a β-barrel domain with 10 β-strands and a signal peptide at the Nterminus. We renamed the gene as an omp1X (outer membrane protein 1 in Xoo), generated its knock out mutant (XooΔomp1X), and compared the protein expression level in the mutant to that in the wild type. A total of 106 proteins displayed more than 1.5-fold difference in expression between the mutant and the wild type strains. COG analysis revealed that these proteins are involved in cell motility as well as signal transduction. In addition, phenotypic analysis demonstrated that motility and biofilm formation in XooΔomp1X are lower than the wild type. These results provide new insights into the functions of outer membrane proteins in Gram-negative bacteria.

Characterization of Thermostable Deblocking Aminopeptidases of Archaeon Thermococcus onnurineus NA1 by Proteomic and Biochemical Approaches: Yeol Gyun Lee , Sun-Hee Leem , Young-Ho Chung , Seung Il Kim; J. Microbiol. 2012;50(5):792-797. Published online November 4, 2012; DOI: https://doi.org/10.1007/s12275-012-2461-2

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Abstract: Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that grows optimally at >80°C. The deblocking aminopeptidase (DAP) (TNA1-DAP1) encoded in Ton_1032 of T. onnurineus NA1 is considered a major DAP. However, four genes encoding putative DAP have been identified from a genomic analysis of T. onnurineus NA1. A proteomic analysis revealed that all four DAPs were differentially induced in YPS culture medium and, particularly, two DAPs (TNA1-DAP1 and TNA1-DAP2) were dominantly expressed in T. onnurineus NA1. The biochemical properties and enzyme activity of DAPs induced in an E. coli expression system suggested that the two major DAPs play complementary roles in T. onnurineus NA1.

Physiological and Metabolic Responses for Hexadecane Degradation in Acinetobacter oleivorans DR1: Jaejoon Jung , Jaemin Noh , Woojun Park; J. Microbiol. 2011;49(2):208-215. Published online May 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0395-8

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31 Citations

Abstract: The hexadecane degradation of Acinetobacter oleivorans DR1 was evaluated with changes in temperature and ionic salt contents. Hexadecane degradation of strain DR1 was reduced markedly by the presence of sodium chloride (but not potassium chloride). High temperature (37°C) was also shown to inhibit the motility, biofilm formation, and hexadecane biodegradation. The biofilm formation of strain DR1 on the oil-water interface might prove to be a critical physiological feature for the degradation of hexadecane. The positive relationship between biofilm formation and hexadecane degradation could be observed at 30°C, but not at low temperatures (25°C). Alterations in cell hydrophobicity and EPS production by temperature and salts were not correlated with biofilm formation and hexadecane degradation. Our proteomic analyses have demonstrated that metabolic changes through the glyoxylate pathway are important for efficient degradation of hexadecane. Proteins involved in fatty acid metabolism, gluconeogenesis, and oxidative stress defense proteins appear to be highly expressed during biodegradation of hexadecane. These results suggested that biofilm formation and oxidative stress defense are important physiological responses for hexadecane degradation along with metabolic switch to glyoxylate pathway in strain DR1.

Proteomic Analysis of Acinetobacter baumannii in Biofilm and Planktonic Growth Mode: Ji-Hyun Shin , Hee-Woo Lee , Sung-Min Kim , Jungmin Kim; J. Microbiol. 2009;47(6):728-735. Published online February 4, 2010; DOI: https://doi.org/10.1007/s12275-009-0158-y

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69 Citations

Abstract: Recently, multidrug-resistant clinical isolates of Acinetobacter baumannii have been found to have a high capacity to form biofilm. It is well known that bacterial cells within biofilms are highly resistant to antibiotics, UV light, acid exposure, dehydration, and phagocytosis in comparison to their planktonic counterparts, which suggests that the cells in a biofilm have altered metabolic activity. To determine which proteins are up-regulated in A. baumannii biofilm cells, we performed a proteomic analysis. A clinical isolate of A. baumannii 1656-2, which was characterized to have a high biofilm forming ability, was cultivated under biofilm and planktonic conditions. Outer membrane enriched A. baumannii 1656-2 proteins were separated by two-dimensional (2-D) gel electrophoresis and the differentially expressed proteins were identified by MALDI-TOF mass spectrometry. The proteins up-regulated or expressed only in biofilm cells of A. baumannii are categorized as follows: (i) proteins processing environmental information such as the outer membrane receptor protein involved in mostly Fe transport, a sensor histidine kinase/response regulator, and diguanylate cyclase (PAS-GGEDF-EAL domain); (ii) proteins involved in metabolism such as NAD- linked malate dehydrogenase, nucleoside-diphosphate sugar epimerase, putative GalE, ProFAR isomerase, and N-acetylmuramoyl-L-alanine amidase; (iii) bacterial antibiotic resistance related proteins; and (iv) proteins related to gene repair such as exodeoxyribonuclease III and GidA. This proteomic analysis provides a fundamental platform for further studies to reveal the role of biofilm in the persistence and tolerance of A. baumannii.

Helicobacter pylori Proteins Response to Nitric Oxide Stress: Wei Qu , Yabin Zhou , Chunhong Shao , Yundong Sun , Qunye Zhang , Chunyan Chen , Jihui Jia; J. Microbiol. 2009;47(4):486-493. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-008-0266-0

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Abstract: Helicobacter pylori is a highly pathogenic microorganism with various strategies to evade human immune responses. Nitric oxide (NO) and reactive nitrogen species (RNS) generated via nitric oxide synthase pathway are important effectors during the innate immune response. However, the mechanisms of H. pylori to survive the nitrosative stress are not clear. Here the proteomic approach has been used to define the adaptive response of H. pylori to nitrosative stress. Proteomic analysis showed that 38 protein spots were regulated by NO donor, sodium nitroprusside (SNP). These proteins were involved in protein processing, antioxidation, general stress response, and virulence, as well as some unknown functions. Particularly, some of them were participated in iron metabolism, potentially under the control of ferric uptake regulator (Fur). Real time PCR revealed that fur was induced under nitrosative stress, consistent with our deduction. One stress-related protein up-regulated under nitrosative conditions was thioredoxin reductase (TrxR). Inactivation of fur or trxR can lead to increased susceptivity to nitrosative stress respectively. These studies described the adaptive response of H. pylori to nitric oxide stress, and analyzed the relevant role of Fur regulon and TrxR in nitrosative stress management.

Note] Comparative Analysis of 2,4,6-Trinitrotoluene (TNT)-Induced Cellular Responses and Proteomes in Pseudomonas sp. HK-6 in Two Types of Media: Yun-Seok Cho , Bheong-Uk Lee , Hyung-Yeel Kahng , Kye-Heon Oh; J. Microbiol. 2009;47(2):220-224. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0108-0

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Abstract: TNT-induced cellular responses and proteomes in Pseudomonas sp. HK-6 were comparatively analyzed in two different media: basal salts (BS) and Luria broth (LB). HK-6 cells could not degrade more than 0.5 mM TNT with BS medium, while in LB medium, they exhibited the enhanced capability to degrade as much as 3.0 mM TNT. Analysis of total cellular fatty acids in HK-6 cells suggested that the relative abundance of several saturated or unsaturated fatty acids is altered under TNT-mediated stress conditions. Scanning electron microscopy showed the presence of perforations, irregular rod formations, and wrinkled extracellular surfaces in cells under TNT stress. Proteomic analysis of soluble protein fractions from HK-6 <br>cultures grown with TNT as a substrate revealed 11 protein spots induced by TNT. Among these, seven proteins (including Alg8, AlgB, NirB, and the AhpC/Tsa family) were detected only in LB medium containing TNT. The proteins AspS, Tsf, and assimilatory nitrate reductase were increasingly expressed only in BS medium containing TNT. The protein dGTPase was found to be induced and expressed when cells were grown in either type of TNT-containing media. These results provide a better understanding of the cytotoxicity and survival mechanism used by Pseudomonas sp. HK-6 when placed under TNT stress conditions.

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