Journal of Microbiology

Volume 59(10); October 2021

Journal Articles

[PROTOCOL] Flow cytometric monitoring of the bacterial phenotypic diversity in aquatic ecosystems: Jin-Kyung Hong , Soo Bin Kim , Seok Hyun Ahn , Yongjoo Choi , Tae Kwon Lee; J. Microbiol. 2021;59(10):879-885. Published online September 23, 2021; DOI: https://doi.org/10.1007/s12275-021-1443-7

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Abstract: Flow cytometry is a promising tool used to identify the phenotypic features of bacterial communities in aquatic ecosystems by measuring the physical and chemical properties of cells based on their light scattering behavior and fluorescence. Compared to molecular or culture-based approaches, flow cytometry is suitable for the online monitoring of microbial water quality because of its relatively simple sample preparation process, rapid analysis time, and high-resolution phenotypic data. Advanced statistical techniques (e.g., denoising and binning) can be utilized to successfully calculate phenotypic diversity by processing the scatter data obtained from flow cytometry. These phenotypic diversities were well correlated with taxonomic-based diversity computed using nextgeneration 16S RNA gene sequencing. The protocol provided in this paper should be a useful guide for a fast and reliable flow cytometric monitoring of bacterial phenotypic diversity in aquatic ecosystems.

Description of Microbacterium luteum sp. nov., Microbacterium cremeum sp. nov., and Microbacterium atlanticum sp. nov., three novel C50 carotenoid producing bacteria: Fuquan Xie , Siwen Niu , Xihuang Lin , Shengxiang Pei , Li Jiang , Yun Tian , Gaiyun Zhang; J. Microbiol. 2021;59(10):886-897. Published online September 7, 2021; DOI: https://doi.org/10.1007/s12275-021-1186-5

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Abstract: We have identified three Microbacterium strains, A18JL200T, NY27T, and WY121T, that produce C50 carotenoids. Taxonomy shows they represent three novel species. These strains shared < 98.5% 16S rRNA gene sequence identity with each other and were closely related to Microbacterium aquimaris JCM 15625T, Microbacterium yannicii JCM 18959T, Microbacterium ureisolvens CFH S00084T, and Microbacterium hibisci CCTCC AB 2016180T. Digital DNA-DNA hybridization (dDDH) values and average nucleotide identity (ANI) showed differences among the three strains and from their closest relatives, with values ranging from 20.4% to 34.6% and 75.5% to 87.6%, respectively. These values are below the threshold for species discrimination. Both morphology and physiology also differed from those of phylogenetically related Microbacterium species, supporting that they are indeed novel species. These strains produce C50 carotenoids (mainly decaprenoxanthin). Among the three novel species, A18JL200T had the highest total yield in carotenoids (6.1 mg/L or 1.2 mg/g dry cell weight). Unusual dual isoprenoid biosynthetic pathways (methylerythritol phosphate and mevalonate pathways) were annotated for strain A18JL200T. In summary, we found strains of the genus Microbacterium that are potential producers of C50 carotenoids, but their genome has to be investigated further.

Brevibacterium limosum sp. nov., Brevibacterium pigmenatum sp. nov., and Brevibacterium atlanticum sp. nov., three novel dye decolorizing actinobacteria isolated from ocean sediments: Shengxiang Pei , Siwen Niu , Fuquan Xie , Wenjing Wang , Shuang Zhang , Gaiyun Zhang; J. Microbiol. 2021;59(10):898-910. Published online September 7, 2021; DOI: https://doi.org/10.1007/s12275-021-1235-0

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Abstract: During a study of the marine actinobacterial biodiversity, a large number of Brevibacterium strains were isolated. Of these, five that have relatively low 16S rRNA gene similarity (98.5– 99.3%) with validly published Brevibacterium species, were chosen to determine taxonomic positions. On the basis of 16S rRNA gene sequence analysis and BOX-PCR fingerprinting, strains o2T, YB235T, and WO024T were selected as representative strains. Genomic analyses, including average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), clearly differentiated the three strains from each other and from their closest relatives, with values ranging from 82.8% to 91.5% for ANI and from 26.7% to 46.5% for dDDH that below the threshold for species delineation. Strains YB235T, WO024T, and o2T all exhibited strong and efficient decolorization activity in congo red (CR) dyes, moderate decolorization activity in toluidine blue (TB) dyes and poor decolorization in reactive blue (RB) dyes. Genes coding for peroxidases and laccases were identified and accounted for these strains’ ability to effectively oxidize a variety of dyes with different chemical structures. Mining of the whole genome for secondary metabolite biosynthesis gene clusters revealed the presence of gene clusters encoding for bacteriocin, ectoine, NRPS, siderophore, T3PKS, terpene, and thiopeptide. Based on the phylogenetic, genotypic and phenotypic data, strains o2T, YB235T and WO024T clearly represent three novel taxa within the genus Brevibacterium, for which the names Brevibacterium limosum sp. nov. (type strain o2T = JCM 33844T = MCCC 1A09961T), Brevibacterium pigmenatum sp. nov. (type strain YB235T = JCM 33843T = MCCC 1A09842T) and Brevibacterium atlanticum sp. nov. (type strain WO024T = JCM 33846T = MCCC 1A16743T) are proposed.

The transcription factor Cas5 suppresses hyphal morphogenesis during yeast-form growth in Candida albicans: Jong-Myeong Kim , Hye Yun Moon , Dong Wook Lee , Hyun Ah Kang , Jeong-Yoon Kim; J. Microbiol. 2021;59(10):911-919. Published online September 7, 2021; DOI: https://doi.org/10.1007/s12275-021-1326-y

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Abstract: Candida albicans is an opportunistic human pathogen that exists as yeast, hyphal or pseudohyphal forms depending on pH, nutrients, and temperature. The morphological transition from yeast to hyphae, which is required for the complete virulence of C. albicans, is controlled by many transcription factors that activate or repress hypha-specific genes. The C. albicans transcriptional factor Cas5, a key regulator of genes involved in cell wall integrity, affects the susceptibility of C. albicans to fluconazole, an inhibitor of ergosterol synthesis. In this study, we found that deletion of CAS5 in C. albicans decreased the expression levels of a set of ergosterol biosynthesis genes, such as ERG2, ERG3, ERG5, ERG6, ERG11, and ERG24,
result
ing in the accumulation of lanosterol and zymosterol, which are intermediate metabolites in the ergosterol biosynthesis pathway. Interestingly, it was observed that the cas5Δ/Δ mutant could not maintain the yeast form under non-hyphainducing conditions, while the CAS5-overexpressing cells could not form hyphae under hypha-inducing conditions. Consistent with these observations, the cas5Δ/Δ mutant highly expressed hypha-specific genes, ALS3, ECE1, and HWP1, under non-hypha-inducing conditions. In addition, CAS5 transcription was significantly downregulated immediately after hyphal initiation in the wild-type strain. Furthermore, the cas5Δ/Δ mutant reduced the transcription of NRG1, which encodes a major repressor of hyphal morphogenesis, while Cas5 overexpression increased the transcription of NRG1 under hyphainducing conditions. Collectively, this study suggests the potential role of Cas5 as a repressor of hypha-specific genes during yeast-form growth of C. albicans.

The small RNA RsaF regulates the expression of secreted virulence factors in Staphylococcus aureus Newman: Niralee Patel , Mrinalini Nair; J. Microbiol. 2021;59(10):920-930. Published online September 23, 2021; DOI: https://doi.org/10.1007/s12275-021-1205-6

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Abstract: The pathogenesis of Staphylococcus aureus, from local infections to systemic dissemination, is mediated by a battery of virulence factors that are regulated by intricate mechanisms, which include regulatory proteins and small RNAs (sRNAs) as key regulatory molecules. We have investigated the involvement of sRNA RsaF, in the regulation of pathogenicity genes hyaluronate lyase (hysA) and serine proteaselike protein D (splD), by employing S. aureus strains with disruption and overexpression of rsaF. Staphylococcus aureus strain with disruption of rsaF exhibited marked down-regulation of hysA transcripts by 0.2 to 0.0002 fold, and hyaluronate lyase activity by 0.2–0.1 fold, as well as increased biofilm formation, during growth from log phase to stationery phase. These mutants also displayed down-regulation of splD transcripts by 0.8 to 0.005 fold, and reduced activity of multiple proteases by zymography. Conversely, overexpression of rsaF resulted in a 2- to 4- fold increase in hysA mRNA levels and hyaluronidase activity. Both hysA and splD mRNAs demonstrated an increased stability in RsaF+ strains. In silico RNA-RNA interaction indicated a direct base pairing of RsaF with hysA and splD mRNAs, which was established in electrophoretic mobility shift assays. The findings demonstrate a positive regulatory role for small RNA RsaF in the expression of the virulence factors, HysA and SplD.

Discovery of novel glycoside hydrolases from C-glycoside-degrading bacteria using sequence similarity network analysis: Bin Wei , Ya-Kun Wang , Jin-Biao Yu , Si-Jia Wang , Yan-Lei Yu , Xue-Wei Xu , Hong Wang; J. Microbiol. 2021;59(10):931-940. Published online September 23, 2021; DOI: https://doi.org/10.1007/s12275-021-1292-4

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Abstract: C-Glycosides are an important type of natural product with significant bioactivities, and the C-glycosidic bonds of C-glycosides can be cleaved by several intestinal bacteria, as exemplified by the human faeces-derived puerarin-degrading bacterium Dorea strain PUE. However, glycoside hydrolases in these bacteria, which may be involved in the C-glycosidic bond cleavage of C-glycosides, remain largely unknown. In this study, the genomes of the closest phylogenetic neighbours of five puerarin-degrading intestinal bacteria (including Dorea strain PUE) were retrieved, and the protein-coding genes in the genomes were subjected to sequence similarity network (SSN) analysis. Only four clusters of genes were annotated as glycoside hydrolases and observed in the genome of D. longicatena DSM 13814T (the closest phylogenetic neighbour of Dorea strain PUE); therefore, genes from D. longicatena DSM 13814T belonging to these clusters were selected to overexpress recombinant proteins (CG1, CG2, CG3, and CG4) in Escherichia coli BL21(DE3). In vitro assays indicated that CG4 efficiently cleaved the O-glycosidic bond of daidzin and showed moderate β-D-glucosidase and β-D-xylosidase activity. CG2 showed weak activity in hydrolyzing daidzin and pNP- β-D-fucopyranoside, while CG3 was identified as a highly selective and efficient α-glycosidase. Interestingly, CG3 and CG4 could be selectively inhibited by daidzein, explaining their different performance in kinetic studies. Molecular docking studies predicted the molecular determinants of CG2, CG3, and CG4 in substrate selectivity and inhibition propensity. The present study identified three novel and distinctive glycoside hydrolases, highlighting the potential of SSN in the discovery of novel enzymes from genomic data.

Linking the gut microbiota to persistent symptoms in survivors of COVID-19 after discharge: Yaya Zhou† , Jianchu Zhang† , Dongmei Zhang , Wan-Li Ma , Xiaorong Wang; J. Microbiol. 2021;59(10):941-948. Published online August 12, 2021; DOI: https://doi.org/10.1007/s12275-021-1206-5

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Abstract: Several follow-up studies have found that COVID-19 (coronavirus disease 2019) patients had persistent symptoms after discharge. Gut microbiota play an important role in human health and immune responses. Therefore, this study investigated the gut microbiota of recovered COVID-19 patients and the correlations between gut microbiota and persistent symptoms after discharge. Stool samples were collected from 15 recovered healthcare workers (HCWs) with COVID-19 at three months after discharge, in addition, stool samples were collected from 14 healthy controls (HCs) to perform 16S rRNA gene sequencing between May and July 2020. Compared with HCs, recovered HCWs had reduced bacterial diversity at three months after discharge, with a significantly higher relative abundance of opportunistic pathogens, and a significantly lower relative abundance of beneficial bacteria. In addition, Escherichia unclassified was positively correlated with persistent symptoms at three months after discharge, including fatigue (r = 0.567, p = 0.028), chest tightness after activity (r = 0.687, p = 0.005), and myalgia (r = 0.523, p = 0.045). Intestinibacter bartlettii was positively correlated with anorexia (r = 0.629, p = 0.012) and fatigue (r = 0.545, p = 0.036). However, Faecalibacterium prausnitzii was negatively correlated with chest tightness after activity (r = -0.591, p = 0.02), and Intestinimonas butyriciproducens was negatively correlated with cough (r = -0.635, p = 0.011). In conclusion, the gut microbiota of recovered HCWs with COVID-19 at three months after discharge was different from that of HCs, and altered gut microbiota was correlated with persistent symptoms after discharge, highlighting that gut microbiota may play an important role in the recovery of patients with COVID-19.

The type II histidine triad protein HtpsC facilitates invasion of epithelial cells by highly virulent Streptococcus suis serotype 2: Yunjun Lu , Shu Li , Xiaodong Shen , Yan Zhao , Dongming Zhou , Dan Hu , Xushen Cai , Lixia Lu , Xiaohui Xiong , Ming Li , Min Cao; J. Microbiol. 2021;59(10):949-957. Published online September 7, 2021; DOI: https://doi.org/10.1007/s12275-021-1129-1

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Abstract: Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen that presents a significant threat both to pigs and to workers in the pork industry. The initial steps of S. suis 2 pathogenesis are unclear. In this study, we found that the type II histidine triad protein HtpsC from the highly virulent Chinese isolate 05ZYH33 is structurally similar to internalin A (InlA) from Listeria monocytogenes, which plays an important role in mediating listerial invasion of epithelial cells. To determine if HtpsC and InlA function similarly, an isogenic htpsC mutant (ΔhtpsC) was generated in S. suis by homologous recombination. The htpsC deletion strain exhibited a diminished ability to adhere to and invade epithelial cells from different sources. Double immunofluorescence microscopy also revealed reduced survival of the ΔhtpsC mutant after cocultivation with epithelium. Adhesion to epithelium and invasion by the wild type strain was inhibited by a monoclonal antibody against E-cadherin. In contrast, the htpsC-deficient mutant was unaffected by the same treatment, suggesting that E-cadherin is the host-cell receptor that interacts with HtpsC and facilitates bacterial internalization. Based on these results, we propose that HtpsC is involved in the process by which S. suis 2 penetrates host epithelial cells, and that this protein is an important virulence factor associated with cell adhesion and invasion.

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