Journal of Microbiology

Volume 60(1); January 2022

Journal Articles

Devosia rhizoryzae sp. nov., and Devosia oryziradicis sp. nov., novel plant growth promoting members of the genus Devosia, isolated from the rhizosphere of rice plants: Geeta Chhetri , Inhyup Kim , Minchung Kang , Jiyoun Kim , Yoonseop So , Taegun Seo; J. Microbiol. 2022;60(1):1-10. Published online November 26, 2021; DOI: https://doi.org/10.1007/s12275-022-1474-8

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Abstract: Two novel Gram-negative, aerobic, asporogenous, motile, rodshaped, orange and white pigmented, designated as LEGU1T and G19T, were isolated from the roots of rice plants, collected from Goyang, South Korea. Phylogenetic analysis based on their 16S rRNA gene sequences revealed that they belonged to the genus Devosia and formed a different lineage and clusters with different members of the genus Devosia. These strains shared common chemotaxonomic features. In particular, they had Q-10 as the sole quinone, phosphatidylglycerol, diphosphatidylglycerol as the principal polar lipids and C16:0, C18:1 ω7c 11-methyl and summed feature 8 (comprising C18:1 ω7c/ C18:1 ω6c) as the main fatty acids. The draft genome sequences of strains LEGU1T and G19T were 3,524,978 and 3,495,520 bp in size, respectively. Their average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were 72.8–81.9% and 18.7–25.1%, respectively, with each other and type strains of related species belonging to the genus Devosia, suggesting that these two strains represent novel species. The G + C content of strains LEGU1T and G19T were 62.1 and 63.8%, respectively. Of the two strains, only LEGU1T produced carotenoid and flexirubin-type pigment. Both strains produced siderophore and indole acetic acid (IAA) in the presence of L-tryptophan. Siderophore biosynthesis genes, auxin responsive genes and tryptophan biosynthesis genes were present in their genomes. The present study aimed to determine the detailed taxonomic positions of the strains using the modern polyphasic approach. Based on the results of polyphasic analysis, these strains are suggested to be two novel bacterial species within the genus Devosia. The proposed names are D. rhizoryzae sp. nov., and Devosia oryziradicis sp. nov., respectively. The plant growth promoting effects of these strains suggest that they can be exploited to improve rice crop productivity. The type strain of D. rhizoryzae is LEGU1T (KCTC 82712T = NBRC 114485T) and D. oryziradicis is G19T (KCTC 82688T = NBRC 114842T).

Constantimarinum furrinae gen. nov., sp. nov., a marine bacterium isolated from saline volcanic rock aquifer (lava seawater) at Jeju Island, Republic of Korea: Sung-Hyun Yang , Hyun-Myung Oh , Mi-Jeong Park , Dongil Jang , Kae Kyoung Kwon; J. Microbiol. 2022;60(1):11-17. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1468-6

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Abstract: A Gram-stain-negative, aerobic, rod-shaped (0.3–0.5 × 1.0– 1.9 μm), non-motile marine bacterium designated as ALE3EIT was isolated from a saline volcanic rock aquifer (lava seawater) on Jeju Island, Republic of Korea. The 16S rRNA gene sequence analysis revealed that strain ALE3EIT showed high similarity to ‘Altibacter lentus’ JLT2010T (97.2%), followed by Marixanthomonas ophiurae KMM 3046T (94.5%). Growth was observed at 10–41°C (optimum, 30°C), at pH 6.0–8.5 (optimum, pH 7.5) and at 0.5–8% (optimum, 4.0%) NaCl. The predominant cellular fatty acids were iso-C15:0 (23.5%), iso-C16:0 (10.2%), iso-C16:0 3OH (10.5%), and iso-C17:0 3OH (16.8%). The DNA G + C contents was 40.4 mol%. The major respiratory quinone was MK-6. The major polar lipids were determined to be phosphatidylethanolamine, two unidentified glycolipids, and two unidentified aminolipids. Several phenotypic characteristics such as production of acetoin, activities of arginine dihydrolase and acid phosphatase, and utilization pattern of carbon sources differentiate strain ALE3EIT from ‘A. lentus’ JLT2010T. Activities of the lipase, trypsin, α- chymotrypsin and gelatinase and utilization pattern of carbon sources differentiate strain ALE3EIT from M. ophiurae KMM 3046T. The genome of strain ALE3EIT is 3.0 Mbp long and its ANI and AAI values against ‘A. lentus’ JLT2010T were 76.58 and 72.76, respectively, however, AAI values against members in other genera were lower than 72%. The phylogenomic tree inferred by PhyloPhlAn clearly differentiated the strain ALE3EIT together with strain JLT2010T from other genera in the Falvobacteriaceae. This polyphasic taxonomic data indicates that strain ALE3EIT should be identified as a novel species in the genus ‘Altibacter’, however, the name has not been validated. Therefore, the strain is classified as a novel genus and is proposed as Constantimarinum furrinae gen. nov., sp. nov. The type strain is ALE3EIT (= KCCM 43303T = JCM 33022T).

Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae: Hye Yun Moon† , Gyu Hun Sim† , Hyeon Jin Kim , Keunpil Kim , Hyun Ah Kang; J. Microbiol. 2022;60(1):18-30. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1580-7

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Abstract: We evaluated the Cre-lox and CRISPR-Cas9 systems as markerrecycling tools in Saccharomyces cerevisiae recombinants containing multiple-integrated expression cassettes. As an initial trial, we constructed rDNA-nontranscribed spacer- or Ty4- based multiple integration vectors containing the URA3 marker flanked by the loxP sequence. Integrants harboring multiple copies of tHMG1 and NNV-CP expression cassettes were obtained and subsequently transformed with the Cre plasmid. However, the simultaneous pop-out of the expression cassettes along with the URA3 marker hampered the use of Cre-lox as a marker-recycling tool in multiple integrants. As an alternative, we constructed a set of CRISPR-Cas9-gRNA vectors containing gRNA targeted to auxotrophic marker genes. Transformation of multiple integrants of tHMG1 and NNV-CP cassettes by the Cas9-gRNA vector in the presence of the URA3 (stop) donor DNA fragments generated the Ura- transformants retaining multiple copies of the expression cassettes. CRISPR-Cas9-based inactivation led to the recycling of the other markers, HIS3, LEU2, and TRP1, without loss of expression cassettes in the recombinants containing multiple copies of tHMG1, NNV-CP, and SfBGL1 cassettes, respectively. Reuse of the same selection marker in marker-inactivated S. cerevisiae was validated by multiple integrations of the TrEGL2 cassette into the S. cerevisiae strain expressing SfBGL1. These results demonstrate that introducing stop codons into selection marker genes using the CRISPR-Cas9 system with donor DNA fragments is an efficient strategy for markerrecycling in multiple integrants. In particular, the continual reuse of auxotrophic markers would facilitate the construction of a yeast cell factory containing multiple copies of expression cassettes without antibiotic resistance genes.

Effects of rehydration on physiological and transcriptional responses of a water-stressed rhizobium: Jie Zhu , Xin Jiang , Dawei Guan , Yaowei Kang , Li Li , Fengming Cao , Baisuo Zhao , Mingchao Ma , Ji Zhao , Jun Li; J. Microbiol. 2022;60(1):31-46. Published online November 26, 2021; DOI: https://doi.org/10.1007/s12275-022-1325-7

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Abstract: As a microsymbiont of soybean, Bradyrhizobium japonicum plays an important role in symbiotic nitrogen fixation and sustainable agriculture. However, the survival of B. japonicum cells under water-deplete (e.g., drought) and water-replete (e.g., flood) conditions is a major concern affecting their nitrogen-fixing ability by establishing the symbiotic relationship with the host. In this study, we isolated a water stress tolerant rhizobium from soybean root nodules and tested its survival under water-deplete conditions. The rhizobium was identified as Bradyrhizobium japonicum and named strain 5038. Interestingly, both plate counting and live/dead fluorescence staining assays indicate that a number of viable but non-culturable cells exist in the culture medium upon the rehydration process which could cause dilution stress. Bradyrhizobium japonicum 5038 cells increased production of exopolysaccharide (EPS) and trehalose when dehydrated, suggesting that protective responses were stimulated. As expected, cells reduced their production upon the subsequent rehydration. To examine differential gene expression of B. japonicum 5038 when exposed to water-deplete and subsequent waterreplete conditions, whole-genome transcriptional analysis was performed under 10% relative humidity (RH), and subsequent 100% RH, respectively. A total of 462 differentially expressed genes (DEGs, > 2.0-fold) were identified under the 10% RH condition, while 3,776 genes showed differential expression during the subsequent rehydration (100% RH) process. Genes involved in signal transduction, inorganic ion transport, energy production and metabolisms of carbohydrates, amino acids, and lipids were far more up-regulated than downregulated in the 10% RH condition. Notably, trehalose biosynthetic genes (otsAB, treS, and treYZ), genes ligD, oprB, and a sigma factor rpoH were significantly induced by 10% RH. Under the subsequent 100% RH condition, genes involved in transcription, translation, cell membrane regulation, replication and repair, and protein processing were highly up-regulated. Interestingly, most of 10%-RH inducible genes displayed rehydration-repressed, except three genes encoding heat shock (Hsp20) proteins. Therefore, this study provides molecular evidence for the switch of gene expression of B. japonicum cells when encountered the opposite water availability from water-deplete to water-replete conditions.

Promoter exchange of the cryptic nonribosomal peptide synthetase gene for oligopeptide production in Aspergillus oryzae: Chanikul Chutrakul , Sarocha Panchanawaporn , Sukanya Jeennor , Jutamas Anantayanon , Kobkul Laoteng; J. Microbiol. 2022;60(1):47-56. Published online November 9, 2021; DOI: https://doi.org/10.1007/s12275-022-1442-3

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Abstract: Oligopeptides with functional activities are of current interest in the nutraceutical and medical sectors. The development of the biosynthetic process of oligopeptides through a nonribosomal peptide synthetase (NRPS) system has become more challenging. To develop a production platform for nonribosomal peptides (NRPs), reprogramming of transcriptional regulation of the acv gene encoded ACV synthetase (ACVS) was implemented in Aspergillus oryzae using the CRISPRCas9 system. Awakening silent acv expression was successfully achieved by promoter substitution. Among the three exchanged promoters, AoPgpdA, AoPtef1, and PtPtoxA, the replacement of the native promoter with AoPgpdA led to the highest ACV production in A. oryzae. However, the ACV production of the AoPGpdA strain was also dependent on the medium composition, in which urea was the best nitrogen source, and a C:N ratio of 20:1 was optimal for tripeptide production. In addition to cell growth, magnesium ions are an essential element for ACV production and might participate in ACVS activity. It was also found that ACV was the growthassociated product of the engineered strain that might be a
result
of constitutive transcriptional control by the AoPgpdA promoter. This study offers a potential strategy for nonribosomal ACV production using the fungal system, which is applicable for redesigning bioactive oligopeptides with industrial relevance.

Interaction between hypoviral-regulated fungal virulence factor laccase3 and small heat shock protein Hsp24 from the chestnut blight fungus Cryphonectria parasitica: Jeesun Chun† , Yo-Han Ko† , Dae-Hyuk Kim; J. Microbiol. 2022;60(1):57-62. Published online November 26, 2021; DOI: https://doi.org/10.1007/s12275-022-1498-0

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Abstract: Laccase3 is an important virulence factor of the fungus Cryphonectria parasitica. Laccase3 gene (lac3) transcription is induced by tannic acid, a group of phenolic compounds found in chestnut trees, and its induction is regulated by the hypovirus CHV1 infection. CpHsp24, a small heat shock protein gene of C. parasitica, plays a determinative role in stress adaptation and pathogen virulence. Having uncovered in our previous study that transcriptional regulation of the CpHsp24 gene in response to tannic acid supplementation and CHV1 infection was similar to that of the lac3, and that conserved phenotypic changes of reduced virulence were observed in mutants of both genes, we inferred that both genes were implicated in a common pathway. Building on this finding, in this paper we examined whether the CpHsp24 protein (CpHSP24) was a molecular chaperone for the lac3 protein (LAC3). Our pull-down experiment indicated that the protein products of the two genes directly interacted with each other. Heterologous co-expression of CpHsp24 and lac3 genes using Saccharomyces cerevisiae resulted in more laccase activity in the cotransformant than in a parental lac3-expresssing yeast strain. These findings suggest that CpHSP24 is, in fact, a molecular chaperone for the LAC3, which is critical component of fungal pathogenesis.

Improved tolerance of recombinant Chlamydomonas rainhardtii with putative 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase from Pyropia yezoensis to nitrogen starvation: Seo-jeong Park , Joon Woo Ahn , Jong-il Choi; J. Microbiol. 2022;60(1):63-69. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1491-7

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Abstract: In a previous study, a putative 2-amino-3-carboxymuconate- 6-semialdehyde decarboxylase (ACMSD) was highly expressed in a mutant strain of Pyropia yezoensis, which exhibited an improved growth rate compared to its wild strain. To investigate the functional role of the putative ACMSD (Pyacmsd) of P. yezoensis, the putative Pyacmsd was cloned and expressed in Chlamydomonas reinhardtii. Recombinant C. reinhardtii cells with Pyacmsd (Cr_Pyacmsd) exhibited enhanced tolerance compared to control C. reinhardtii cells (Cr_control) under nitrogen starvation. Notably, Cr_Pyacmsd cells showed accumulation of lipids in nitrogen-enriched conditions. These
results
demonstrate the role of Pyacmsd in the generation of acetyl-coenzyme A. Thus, it can be used to enhance the production of biofuel using microalgae such as C. reinhardtii and increase the tolerance of other biological systems to nitrogendeficient conditions.

Direct current exerts electricidal and bioelectric effects on Porphyromonas gingivalis biofilms partially via promoting oxidative stress and antibiotic transport: Peihui Zou , Peng Li , Jia Liu , Pei Cao , Qingxian Luan; J. Microbiol. 2022;60(1):70-78. Published online November 26, 2021; DOI: https://doi.org/10.1007/s12275-022-1238-5

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Abstract: Low electric current can inhibit certain microbial biofilms and enhance the efficacy of antimicrobials against them. This study investigated the electricidal and bioelectric effects of direct current (DC) against Porphyromonas gingivalis biofilms as well as the underlying mechanisms. Here, we firstly showed that DC significantly suppressed biofilm formation of P. gingivalis in time- and intensity-dependent manners, and markedly inhibited preformed P. gingivalis biofilms. Moreover, DC enhanced the killing efficacy of metronidazole (MTZ) and amoxicillin with clavulanate potassium (AMC) against the biofilms. Notably, DC-treated biofilms displayed upregulated intracellular ROS and expression of ROS related genes (sod, feoB, and oxyR) as well as porin gene. Interestingly, DC-induced killing of biofilms was partially reversed by ROS scavenger N-dimethylthiourea (DMTU), and the synergistic effect of DC with MTZ/AMC was weakened by small interfering RNA of porin gene (si-Porin). In conclusion, DC can exert electricidal and bioelectric effects against P. gingivalis biofilms partially via promotion of oxidative stress and antibiotic transport, which offers a promising approach for effective management of periodontitis.

Gene deletion and constitutive expression of the pectate lyase gene 1 (MoPL1) lead to diminished virulence of Magnaporthe oryzae: Alex Wegner , Florencia Casanova , Marco Loehrer , Angelina Jordine , Stefan Bohnert , Xinyu Liu , Zhengguang Zhang , Ulrich Schaffrath; J. Microbiol. 2022;60(1):79-88. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1074-7

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Abstract: Phytopathogenic fungi are known to secrete specific proteins which act as virulence factors and promote host colonization. Some of them are enzymes with plant cell wall degradation capability, like pectate lyases (Pls). In this work, we examined the involvement of Pls in the infection process of Magnaporthe oryzae, the causal agent of rice blast disease. From three Plgenes annotated in the M. oryzae genome, only transcripts of MoPL1 considerably accumulated during the infection process with a peak at 72 h post inoculation. Both, gene deletion and a constitutive expression of MoPL1 in M. oryzae led to a significant reduction in virulence. By contrast, mutants that constitutively expressed an enzymatic inactive version of MoPl1 did not differ in virulence compared to the wild type isolate. This indicates that the enzymatic activity of MoPl1 is responsible for diminished virulence, which is presumably due to degradation products recognized as danger associated molecular patterns (DAMPs), which strengthen the plant immune response. Microscopic analysis of infection sites pointed to an increased plant defense response. Additionally, MoPl1 tagged with mRFP, and not the enzymatic inactive version, focally accumulated in attacked plant cells beneath appressoria and at sites where fungal hyphae transverse from one to another cell. These findings shed new light on the role of pectate lyases during tissue colonization in the necrotrophic stage of M. oryzae's life cycle.

Potent antibacterial and antibiofilm activities of TICbf-14, a peptide with increased stability against trypsin: Liping Wang , Xiaoyun Liu , Xinyue Ye , Chenyu Zhou , Wenxuan Zhao , Changlin Zhou , Lingman Ma; J. Microbiol. 2022;60(1):89-99. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1368-9

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Abstract: The poor stability of peptides against trypsin largely limits their development as potential antibacterial agents. Here, to obtain a peptide with increased trypsin stability and potent antibacterial activity, TICbf-14 derived from the cationic peptide Cbf-14 was designed by the addition of disulfide-bridged hendecapeptide (CWTKSIPPKPC) loop. Subsequently, the trypsin stability and antimicrobial and antibiofilm activities of this peptide were evaluated. The possible mechanisms underlying its mode of action were also clarified. The results showed that TICbf-14 exhibited elevated trypsin inhibitory activity and effectively mitigated lung histopathological damage in bacteria-infected mice by reducing the bacterial counts, further inhibiting the systemic dissemination of bacteria and host inflammation. Additionally, TICbf-14 significantly repressed bacterial swimming motility and notably inhibited biofilm formation. Considering the mode of action, we observed that TICbf-14 exhibited a potent membrane-disruptive mechanism, which was attributable to its destructive effect on ionic bridges between divalent cations and LPS of the bacterial membrane. Overall, TICbf-14, a bifunctional peptide with both antimicrobial and trypsin inhibitory activity, is highly likely to become an ideal candidate for drug development against bacteria.

Lactobacillus plantarum-derived metabolites sensitize the tumorsuppressive effects of butyrate by regulating the functional expression of SMCT1 in 5-FU-resistant colorectal cancer cells: Hye-Ju Kim , JaeJin An , Eun-Mi Ha; J. Microbiol. 2022;60(1):100-117. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1533-1

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26 Citations

Abstract: A critical obstacle to the successful treatment of colorectal cancer (CRC) is chemoresistance. Chemoresistant CRC cells contribute to treatment failure by providing a mechanism of drug lethargy and modifying chemoresistance-associated molecules. The gut microbiota provide prophylactic and therapeutic effects by targeting CRC through anticancer mechanisms. Among them, Lactobacillus plantarum contributes to the health of the host and is clinically effective in treating CRC. This study confirmed that 5-fluorouracil (5-FU)-resistant CRC HCT116 (HCT116/5FUR) cells acquired butyrateinsensitive properties. To date, the relationship between 5- FU-resistant CRC and butyrate resistance has not been elucidated. Here, we demonstrated that the acquisition of butyrate resistance in HCT116/5FUR cells was strongly correlated with the inhibition of the expression and function of SMCT1, a major transporter of butyrate in colonocytes. L. plantarum-cultured cell-free supernatant (LP) restored the functional expression of SMCT1 in HCT116/5FUR cells, leading to butyrate-induced antiproliferative effect and apoptosis. These results suggest that LP has a synergistic effect on the SMCT1/butyrate-mediated tumor suppressor function and is a potential chemosensitizer to overcome dual 5-FU and butyrate resistance in HCT116 cells.

The human symbiont Bacteroides thetaiotaomicron promotes diet-induced obesity by regulating host lipid metabolism: Sang-Hyun Cho , Yong-Joon Cho , Joo-Hong Park; J. Microbiol. 2022;60(1):118-127. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1614-1

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Abstract: The gut microbiome plays an important role in lipid metabolism. Consumption of a high-fat diet (HFD) alters the bacterial communities in the gut, leading to metabolic disorders. Several bacterial species have been associated with diet-induced obesity, nonalcoholic fatty liver disease, and metabolic syndrome. However, the mechanisms underlying the control of lipid metabolism by symbiotic bacteria remain elusive. Here, we show that the human symbiont Bacteroides thetaiotaomicron aggravates metabolic disorders by promoting lipid digestion and absorption. Administration of B. thetaiotaomicron to HFD-fed mice promoted weight gain, elevated fasting glucose levels, and impaired glucose tolerance. Furthermore, B. thetaiotaomicron treatment upregulated the gene expression of the fatty acid transporter and increased fatty acid accumulation in the liver. B. thetaiotaomicron inhibits expression of the gene encoding a lipoprotein lipase inhibitor, angiopoietin-like protein 4 (ANGPTL4), thereby increasing lipase activity in the small intestine. In particular, we found that B. thetaiotaomicron induced the expression of hepcidin, the master regulator of iron metabolism and an antimicrobial peptide, in the liver. Hepcidin treatment resulted in a decrease in ANGPTL4 expression in Caco-2 cells, whereas treatment with an iron chelator restored ANGPTL4 expression in hepcidin- treated cells. These results indicate that B. thetaiotaomicron- mediated regulation of iron storage in intestinal epithelial cells may contribute to increased fat deposition and impaired glucose tolerance in HFD-fed mice.

Gold nanoparticle-DNA aptamer-assisted delivery of antimicrobial peptide effectively inhibits Acinetobacter baumannii infection in mice: Jaeyeong Park , Eunkyoung Shin , Ji-Hyun Yeom , Younkyung Choi , Minju Joo , Minho Lee , Je Hyeong Kim , Jeehyeon Bae , Kangseok Lee; J. Microbiol. 2022;60(1):128-136. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1620-3

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Abstract: Acinetobacter baumannii causes multidrug resistance, leading to fatal infections in humans. In this study, we showed that Lys AB2 P3-His–a hexahistidine-tagged form of an antimicrobial peptide (AMP) loaded onto DNA aptamer-functionalized gold nanoparticles (AuNP-Apt)–can effectively inhibit A. baumannii infection in mice. When A. baumannii-infected mice were intraperitoneally injected with AuNP-Apt loaded with Lys AB2 P3-His, a marked reduction in A. baumannii colonization was observed in the mouse organs, leading to prominently increased survival time and rate of the mice compared to those of the control mice treated with AuNP-Apt or Lys AB2 P3-His only. This study shows that AMPs loaded onto AuNP-Apt could be an effective therapeutic tool against infections caused by multidrug-resistant pathogenic bacteria in humans.

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