A Marine Bacterium with Animal-Pathogen-Like Type III Secretion Elicits the Nonhost Hypersensitive Response in a Land Plant.

Active plant immune response involving programmed cell death called the hypersensitive response (HR) is elicited by microbial effectors delivered through the type III secretion system (T3SS). The marine bacterium Hahella chejuensis contains two T3SSs that are similar to those of animal pathogens, but it was able to elicit HR-like cell death in the land plant Nicotiana benthamiana. The cell death was comparable with the transcriptional patterns of H. chejuensis T3SS-1 genes, was mediated by SGT1, a general regulator of plant resistance, and was suppressed by AvrPto1, a type III-secreted effector of a plant pathogen that inhibits HR. Thus, type III-secreted effectors of a marine bacterium are capable of inducing the nonhost HR in a land plant it has never encountered before. This suggests that plants may have evolved to cope with a potential threat posed by alien pathogen effectors. Our work documents an exceptional case of nonhost HR and provides an expanded perspective for studying plant nonhost resistance.

Gut microbiota of the young ameliorates physical fitness of the aged in mice.

BACKGROUND: Aging is a natural process that an organism gradually loses its physical fitness and functionality. Great efforts have been made to understand and intervene in this deteriorating process. The gut microbiota affects host physiology, and dysbiosis of the microbial community often underlies the pathogenesis of host disorders. The commensal microbiota also changes with aging; however, the interplay between the microbiota and host aging remains largely unexplored. Here, we systematically examined the ameliorating effects of the gut microbiota derived from the young on the physiology and phenotypes of the aged. RESULTS: As the fecal microbiota was transplanted from young mice at 5 weeks after birth into 12-month-old ones, the thickness of the muscle fiber and grip strength were increased, and the water retention ability of the skin was enhanced with thickened stratum corneum. Muscle thickness was also marginally increased in 25-month-old mice after transferring the gut microbiota from the young. Bacteria enriched in 12-month-old mice that received the young-derived microbiota significantly correlated with the improved host fitness and altered gene expression. In the dermis of these mice, transcription of Dbn1 was most upregulated and DBN1-expressing cells increased twice. Dbn1-heterozygous mice exhibited impaired skin barrier function and hydration. CONCLUSIONS: We revealed that the young-derived gut microbiota rejuvenates the physical fitness of the aged by altering the microbial composition of the gut and gene expression in muscle and skin. Dbn1, for the first time, was found to be induced by the young microbiota and to modulate skin hydration. Our results provide solid evidence that the gut microbiota from the young improves the vitality of the aged. Video Abstract.

Enterotypical Prevotella and three novel bacterial biomarkers in preoperative stool predict the clinical outcome of colorectal cancer.

BACKGROUND: A significant proportion of colorectal cancer (CRC) patients suffer from early recurrence and progression after surgical treatment. Although the gut microbiota is considered as a key player in the initiation and progression of CRC, most prospective studies have been focused on a particular pathobionts such as Fusobacterium nucleatum. Here, we aimed to identify novel prognostic bacteria for CRC by examining the preoperative gut microbiota through 16S ribosomal RNA gene sequencing. RESULTS: We collected stool samples from 333 patients with primary CRC within 2 weeks before surgery and followed up the patients for a median of 27.6 months for progression and 43.6 months for survival. The sequence and prognosis data were assessed using the log-rank test and multivariate Cox proportional hazard analysis. The gut microbiota was associated with the clinical outcomes of CRC patients (Pprogress = 0.011, Pdecease = 0.007). In particular, the high abundance of Prevotella, a representative genus of human enterotypes, indicated lower risks of CRC progression (P = 0.026) and decease (P = 0.0056), while the occurrence of Alistipes assigned to Bacteroides sp., Pyramidobacter piscolens, Dialister invisus, and Fusobacterium nucleatum indicated a high risk of progression. A microbiota-derived hazard score considering the five prognostic bacteria accurately predicted CRC progression in 1000 random subsamples; it outperformed widely accepted clinical biomarkers such as carcinoembryonic antigen and lymphatic invasion, after adjustment for the clinicopathological stage (adjusted HR 2.07 [95% CI, 1.61-2.64], P = 7.8e-9, C-index = 0.78). PICRUSt2 suggested that microbial pathways pertaining to thiamine salvage and L-histidine degradation underlie the different prognoses. CONCLUSIONS: The enterotypical genus Prevotella was demonstrated to be useful in improving CRC prognosis, and combined with the four pathobionts, our hazard score based on the gut microbiota should provide an important asset in predicting medical outcomes for CRC patients. Video Abstract.

Transcriptional Potential Determines the Adaptability of Escherichia coli Strains with Different Fitness Backgrounds.

Adaptation through the fitness landscape may be influenced by the gene pool or expression network. However, genetic factors that determine the contribution of beneficial mutations during adaptive evolution are poorly understood. In this study, we experimentally evolved wild-type Escherichia coli K-12 MG1655 and its isogenic derivative that has two additional replication origins and shows higher background fitness. During the short time of experimental evolution, the fitness gains of the two E. coli strains with different fitness backgrounds converged. Populational genome sequencing revealed various mutations with different allele frequencies in evolved populations. Several mutations occurred in genes affecting transcriptional regulation (e.g., RNA polymerase subunit, RNase, ppGpp synthetase, and transcription termination/antitermination factor genes). When we introduced mutations into the ancestral E. coli strains, beneficial effects tended to be lower in the ancestor with higher initial fitness. Replication rate analysis showed that the various replication indices do not correlate with the growth rate. Transcriptome profiling showed that gene expression and gene ontology are markedly enriched in populations with lower background fitness after experimental evolution. Further, the degree of transcriptional change was proportional to the fitness gain. Thus, the evolutionary trajectories of bacteria with different fitness backgrounds can be complex and counterintuitive. Notably, transcriptional change is a major contributor to adaptability. IMPORTANCE Predicting the adaptive potential of bacterial populations can be difficult due to their complexity and dynamic environmental conditions. Also, epistatic interaction between mutations affects the adaptive trajectory. Nevertheless, next-generation sequencing sheds light on understanding evolutionary dynamics through high-throughput genome and transcriptome information. Experimental evolution of two E. coli strains with different background fitness showed that the trajectories of fitness gain, which slowed down during the later stages of evolution, became convergent. This suggests that the adaptability of bacteria can be counterintuitive and that predicting the evolutionary path of bacteria can be difficult even in a constant environment. In addition, transcriptional change is associated with fitness gain during the evolutionary process. Thus, the adaptability of cells depends on their intrinsic genetic capacity for a given evolutionary period. This should be considered when genetically engineered bacteria are optimized through adaptive evolution.

Escherichia coli cell factories with altered chromosomal replication scenarios exhibit accelerated growth and rapid biomass production.

BACKGROUND: Generally, bacteria have a circular genome with a single replication origin for each replicon, whereas archaea and eukaryotes can have multiple replication origins in a single chromosome. In Escherichia coli, bidirectional DNA replication is initiated at the origin of replication (oriC) and arrested by the 10 termination sites (terA-J). RESULTS: We constructed E. coli derivatives with additional or ectopic replication origins, which demonstrate the relationship between DNA replication and cell physiology. The cultures of E. coli derivatives with multiple replication origins contained an increased fraction of replicating chromosomes and the cells varied in size. Without the original oriC, E. coli derivatives with double ectopic replication origins manifested impaired growth irrespective of growth conditions and enhanced cell size, and exhibited excessive and asynchronous replication initiation. The generation time of an E. coli strain with three replication origins decreased in a minimal medium supplemented with glucose as the sole carbon source. As well as cell growth, the introduction of additional replication origins promoted increased biomass production. CONCLUSIONS: Balanced cell growth and physiological stability of E. coli under rapid growth condition are affected by changes in the position and number of replication origins. Additionally, we show that, for the first time to our knowledge, the introduction of replication initiation sites to the chromosome promotes cell growth and increases protein production.

Human gastric microbiota transplantation recapitulates premalignant lesions in germ-free mice.

OBJECTIVE: Gastric cancer (GC) is a leading cause of cancer-related mortality. Although microbes besides Helicobacter pylori may also contribute to gastric carcinogenesis, wild-type germ-free (GF) mouse models investigating the role of human gastric microbiota in the process are not yet available. We aimed to evaluate the histopathological features of GF mouse stomachs transplanted with gastric microbiota from patients with different gastric disease states and their relationships with the microbiota. DESIGN: Microbiota profiles in corpus and antrum tissues and gastric fluid from 12 patients with gastric dysplasia or GC were analysed. Thereafter, biopsied corpus and antrum tissues and gastric fluid from patients (n=15 and n=12, respectively) with chronic superficial gastritis, intestinal metaplasia or GC were inoculated into 42 GF C57BL/6 mice. The gastric microbiota was analysed by amplicon sequencing. Histopathological features of mouse stomachs were analysed immunohistochemically at 1 month after inoculation. An independent set of an additional 15 GF mice was also analysed at 1 year. RESULTS: The microbial community structures of patients with dysplasia or GC in the corpus and antrum were similar. The gastric microbiota from patients with intestinal metaplasia or GC selectively colonised the mouse stomachs and induced premalignant lesions: loss of parietal cells and increases in inflammation foci, in F4/80 and Ki-67 expression, and in CD44v9/GSII lectin expression. Marked dysplastic changes were noted at 1 year post inoculation. CONCLUSION: Major histopathological features of premalignant changes are reproducible in GF mice transplanted with gastric microbiota from patients with intestinal metaplasia or GC. Our results suggest that GF mice are useful for analysing the causality of associations reported in human gastric microbiome studies.

Genome information of the cellulolytic soil actinobacterium Isoptericola dokdonensis DS-3 and comparative genomic analysis of the genus Isoptericola.

The actinobacterial group is regarded as a reservoir of biologically active natural products and hydrolytic enzymes with the potential for biomedical and industrial applications. Here, we present the complete genome sequence of Isoptericola dokdonensis DS-3 isolated from soil in Dokdo, small islets in the East Sea of Korea. This actinomycete harbors a large number of genes encoding carbohydrate-degrading enzymes, and its activity to degrade carboxymethyl cellulose into glucose was experimentally evaluated. Since the genus Isoptericola was proposed after reclassification based on phylogenetic analysis, strains of Isoptericola have been continuously isolated from diverse environments and the importance of this genus in the ecosystem has been suggested by recent culturomic or metagenomic studies. The phylogenic relationships of the genus tended to be closer among strains that had been isolated from similar habitats. By analyzing the properties of published genome sequences of seven defined species in the genus, a large number of genes for carbohydrate hydrolysis and utilization, as well as several biosynthetic gene clusters for secondary metabolites, were identified. Genomic information of I. dokdonensis DS-3 together with comparative analysis of the genomes of Isoptericola provides insights into understanding this actinobacterial group with a potential for industrial applications.

Flavobacterium dauae sp. nov., isolated from rhizosphere soil of a tomato plant.

A bacterial strain, designated TCH3-2T, was isolated from the rhizosphere of tomato plant grown at Dong-A University Agricultural Experiment Station, Republic of Korea. The strain was Gram-stain-negative, obligate aerobic, orange yellow-coloured, motile by gliding and short rod-shaped. Strain TCH3-2 T only grew on 1/2 tryptic soy agar and Luria-Bertani agar among the media tested, with optimum growth at 28 °C and pH 7. Salt of 1 % NaCl was necessary to support the growth of TCH3-2T. Strain TCH3-2T produced flexirubin-type pigments. The predominant cellular fatty acids were iso-C15 : 0 (55.6 %), iso-C17 : 0 3-OH (17.9 %), summed feature 9 (comprising C16 : 0 10-methyl and/or iso-C17 : 1 ω9c; 10.5 %), iso-C15 : 0 3-OH (4.8 %) and anteiso-C15 : 0 (2.3 %). The major menaquinone was menaquinone-6 and the major polar lipids were phosphatidylethanolamine, five unknown aminolipids and three unknown lipids. Phylogenetic analysis based on 16S rRNA sequences indicated that TCH3-2T was closely related to Flavobacterium ummariense DS-12T (95.16 %), Flavobacterium marinum SW105T (95.14 %) and Flavobacterium viscosus YIM 102796T (94.54 %). The draft genome of TCH3-2T comprised ca. 2.8 Mb with a G+C content of 34.61 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between TCH3-2T and closely related Flavobacterium species showed that it belongs to a distinct species. Furthermore, the results of morphological, physiological and biochemical tests allowed further phenotypic differentiation of TCH3-2T from its closest relatives. Thus, chemotaxonomic characteristics together with phylogenetic affiliation illustrate that TCH3-2T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium dauae sp. nov. (type strain TCH3-2T=KACC 19054T=JCM 34025T) is proposed.

A synthetic probiotic engineered for colorectal cancer therapy modulates gut microbiota.

BACKGROUND: Successful chemoprevention or chemotherapy is achieved through targeted delivery of prophylactic agents during initial phases of carcinogenesis or therapeutic agents to malignant tumors. Bacteria can be used as anticancer agents, but efforts to utilize attenuated pathogenic bacteria suffer from the risk of toxicity or infection. Lactic acid bacteria are safe to eat and often confer health benefits, making them ideal candidates for live vehicles engineered to deliver anticancer drugs. RESULTS: In this study, we developed an effective bacterial drug delivery system for colorectal cancer (CRC) therapy using the lactic acid bacterium Pediococcus pentosaceus. It is equipped with dual gene cassettes driven by a strong inducible promoter that encode the therapeutic protein P8 fused to a secretion signal peptide and a complementation system. In an inducible CRC cell-derived xenograft mouse model, our synthetic probiotic significantly reduced tumor volume and inhibited tumor growth relative to the control. Mice with colitis-associated CRC induced by azoxymethane and dextran sodium sulfate exhibited polyp regression and recovered taxonomic diversity when the engineered bacterium was orally administered. Further, the synthetic probiotic modulated gut microbiota and alleviated the chemically induced dysbiosis. Correlation analysis demonstrated that specific bacterial taxa potentially associated with eubiosis or dysbiosis, such as Akkermansia or Turicibacter, have positive or negative relationships with other microbial members. CONCLUSIONS: Taken together, our work illustrates that an effective and stable synthetic probiotic composed of P. pentosaceus and the P8 therapeutic protein can reduce CRC and contribute to rebiosis, and the validity and feasibility of cell-based designer biopharmaceuticals for both treating CRC and ameliorating impaired microbiota. Video abstract.

Genome Analysis of Erwinia amylovora Strains Responsible for a Fire Blight Outbreak in Korea.

Erwinia amylovora is a plant-pathogenic bacterium that causes fire blight disease in Rosaceae plants. Since fire blight is highly contagious and results in serious losses once introduced, it is regulated as a quarantine disease. Recently, for the first time in East Asia, fire blight emerged in Korea with strains of E. amylovora being isolated from lesions of infected trees. Five of those strains were selected and subjected to whole-genome shotgun sequencing. Each strain had two circular replicons, a 3.8-Mb chromosome and a 28-kb plasmid. The genome sequences were compared with those of other E. amylovora strains isolated from different hosts or geographical regions. Genome synteny was analyzed and sequence variations including nucleotide substitutions, inversions, insertions, and deletions were detected. Analysis of the population genomic structure revealed that the five strains form a distinct structural group. Phylogenomic analysis was performed to infer the evolutionary relationships among E. amylovora strains, which indicated that the Korean isolates, all descended from a common ancestor, are closely related to a lineage of North American strains. These results provide useful information for understanding the genomic dynamics of E. amylovora strains including those in Korea, developing genetic markers for surveillance of the pathogen or diagnosis of the disease, and eventually developing measures to eradicate it.

Reverse Engineering Targets for Recombinant Protein Production in Corynebacterium glutamicum Inspired by a Fast-Growing Evolved Descendant.

We previously reported a Corynebacterium glutamicum JH41 strain with a 58% faster growth rate through application of adaptive laboratory evolution. To verify that the fast-reproducing strain was useful as a host for recombinant protein expression, we introduced a plasmid responsible for the secretory production of a recombinant protein. The JH41 strain harboring the plasmid indeed produced the secretory recombinant protein at a 2.7-fold greater rate than its ancestral strain. To provide the reverse engineering targets responsible for boosting recombinant protein production and cell reproduction, we compared the genome sequence of the JH41 strain with its ancestral strain. Among the 15 genomic variations, a point mutation was confirmed in the 14 bases upstream of NCgl1959 (encoding a presumed siderophore-binding protein). This mutation allowed derepression of NCgl1959, thereby increasing iron consumption and ATP generation. A point mutation in the structural gene ramA (A239G), a LuxR-type global transcription regulator involved in central metabolism, allowed an increase in glucose consumption. Therefore, mutations to increase the iron and carbon consumption were concluded as being responsible for the enhanced production of recombinant protein and cell reproduction in the evolved host.

Molecular changes associated with spontaneous phenotypic variation of Paenibacillus polymyxa, a commonly used biocontrol agent, and temperature-dependent control of variation.

There has been a growing interest in deploying plant growth-promoting rhizobacteria (PGPR) as a biological control agent (BCA) to reduce the use of agrochemicals. Spontaneous phenotypic variation of PGPR, which causes the loss of traits crucial for biocontrol, presents a large obstacle in producing commercial biocontrol products. Here, we report molecular changes associated with phenotypic variation in Paenibacillus polymyxa, a PGPR widely used for biocontrol worldwide, and a simple cultural change that can prevent the variation. Compared to B-type (non-variant) cells of P. polymyxa strain E681, its phenotypic variant, termed as F-type, fails to form spores, does not confer plant growth-promoting effect, and displays altered colony and cell morphology, motility, antagonism against other microbes, and biofilm formation. This variation was observed in all tested strains of P. polymyxa, but the frequency varied among them. RNA-seq analysis revealed differential regulation of many genes involved in sporulation, flagella synthesis, carbohydrate metabolism, and antimicrobial production in F-type cells, consistent with their pleiotropic phenotypic changes. F-type cells's sporulation was arrested at stage 0, and the key sporulation gene spo0A was upregulated only in B-type cells. The phenotypic variation could be prevented by altering the temperature for growth. When E681 was cultured at 20 °C or lower, it exhibited no variation for 7 days and still reached ~ 108 cfu/mL, the level sufficient for commercial-scale production of biocontrol products.

Alterations in Gastric Microbial Communities Are Associated with Risk of Gastric Cancer in a Korean Population: A Case-Control Study.

Although the microbiome has a potential role in gastric cancer (GC), little is known about microbial dysbiosis and its functions. This study aimed to observe the associations between the alterations in gastric microbial communities and GC risk. The study participants included 268 GC patients and 288 controls. The 16S rRNA gene sequencing was performed to characterize the microbiome. Streptococcus_NCVM and Prevotella melaninogenica species were highly enriched in cases and controls, respectively. Those who were in the third tertile of P. melaninogenica showed a significantly decreased risk of GC in total (odds ratio (OR): 0.91, 95% confidence interval (CI): 0.38-0.96, p-trend = 0.071). Class Bacilli was phylogenetically enriched in cases, while phylum Actinobacteria, class Actinobacteria were related to the controls. The microbial dysbiosis index (MDI) was significantly higher for the cases compared with the healthy controls in the female population (p = 0.002). Females in the third tertile of the MDI showed a significantly increased risk of GC (OR: 2.66, 95% CI: 1.19-5.99, p-trend = 0.017). Secondary bile acid synthesis and biosynthesis of ansamycins pathways were highly abundant in cases and controls, respectively. Dysbiosis of gastric microbial communities is associated with an increased risk of GC specifically in females.

Accelerated Growth of Corynebacterium glutamicum by Up-Regulating Stress- Responsive Genes Based on Transcriptome Analysis of a Fast-Doubling Evolved Strain.

Corynebacterium glutamicum, an important industrial strain, has a relatively slower reproduction rate. To acquire a growth-boosted C. glutamicum, a descendant strain was isolated from a continuous culture after 600 generations. The isolated descendant C. glutamicum, JH41 strain, was able to double 58% faster (td=1.15 h) than the parental type strain (PT, td=1.82 h). To understand the factors boosting reproduction, the transcriptomes of JH41 and PT strains were compared. The mRNAs involved in respiration and TCA cycle were upregulated. The intracellular ATP of the JH41 strain was 50% greater than the PT strain. The upregulation of NCgl1610 operon (a putative dyp-type heme peroxidase, a putative copper chaperone, and a putative copper importer) that presumed to role in the assembly and redox control of cytochrome c oxidase was found in the JH41 transcriptome. Plasmid-driven expression of the operon enabled the PT strain to double 19% faster (td=1.82 h) than its control (td=2.17 h) with 14% greater activity of cytochrome c oxidase and 27% greater intracellular ATP under the oxidative stress conditions. Upregulations of genes those might enhance translation fitness were also found in the JH41 transcriptome. Plasmid-driven expressions of NCgl0171 (encoding a cold-shock protein) and NCgl2435 (encoding a putative peptidyl-tRNA hydrolase) enabled the PT to double 22% and 32% faster than its control, respectively (empty vector: td=1.93 h, CspA: td=1.58 h, and Pth: td=1.44 h). Based on the results, the factors boosting growth rate in C. gluctamicum were further discussed in the viewpoints of cellular energy state, oxidative stress management, and translation.

Omega Rhodopsins: A Versatile Class of Microbial Rhodopsins.

Microbial rhodopsins are a superfamily of photoactive membrane proteins with covalently bound retinal cofactor. Isomerization of the retinal chromophore upon absorption of a photon triggers conformational changes of the protein to function as ion pumps or sensors. After the discovery of proteorhodopsin in an uncultivated γ-proteobacterium, light-activated proton pumps have been widely detected among marine bacteria and, together with chlorophyll-based photosynthesis, are considered as an important axis responsible for primary production in the biosphere. Rhodopsins and related proteins show a high level of phylogenetic diversity; we focus on a specific class of bacterial rhodopsins containing the 3 omega motif. This motif forms a stack of three nonconsecutive aromatic amino acids that correlates with the B-C loop orientation, and is shared among the phylogenetically close ion pumps such as the NDQ motif-containing sodium-pumping rhodopsin, the NTQ motif-containing chloride-pumping rhodopsin, and some proton-pumping rhodopsins including xanthorhodopsin. Here, we reviewed the recent research progress on these omega rhodopsins, and speculated on their evolutionary origin of functional diversity..

Bioinformatics services for analyzing massive genomic datasets.

The explosive growth of next-generation sequencing data has resulted in ultra-large-scale datasets and ensuing computational problems. In Korea, the amount of genomic data has been increasing rapidly in the recent years. Leveraging these big data requires researchers to use large-scale computational resources and analysis pipelines. A promising solution for addressing this computational challenge is cloud computing, where CPUs, memory, storage, and programs are accessible in the form of virtual machines. Here, we present a cloud computing-based system, Bio-Express, that provides user-friendly, cost-effective analysis of massive genomic datasets. Bio-Express is loaded with predefined multi-omics data analysis pipelines, which are divided into genome, transcriptome, epigenome, and metagenome pipelines. Users can employ predefined pipelines or create a new pipeline for analyzing their own omics data. We also developed several web-based services for facilitating downstream analysis of genome data. Bio-Express web service is freely available at https://www.bioexpress.re.kr/.

Makes caterpillars floppy-like effector-containing MARTX toxins require host ADP-ribosylation factor (ARF) proteins for systemic pathogenicity.

Upon invading target cells, multifunctional autoprocessing repeats-in-toxin (MARTX) toxins secreted by bacterial pathogens release their disease-related modularly structured effector domains. However, it is unclear how a diverse repertoire of effector domains within these toxins are processed and activated. Here, we report that Makes caterpillars floppy-like effector (MCF)-containing MARTX toxins require ubiquitous ADP-ribosylation factor (ARF) proteins for processing and activation of intermediate effector modules, which localize in different subcellular compartments following limited processing of holo effector modules by the internal cysteine protease. Effector domains structured tandemly with MCF in intermediate modules become disengaged and fully activated by MCF, which aggressively interacts with ARF proteins present at the same location as intermediate modules and is converted allosterically into a catalytically competent protease. MCF-mediated effector processing leads ultimately to severe virulence in mice via an MCF-mediated ARF switching mechanism across subcellular compartments. This work provides insight into how bacteria take advantage of host systems to induce systemic pathogenicity.