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Acta Biochimica Et Biophysica Sinica Apr 2021The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (Cas9)-based genome editing tool pCas/pTargetF system that we established...
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (Cas9)-based genome editing tool pCas/pTargetF system that we established previously has been widely used in Escherichia coli MG1655. However, this system failed to manipulate the genome of E. coli BL21(DE3), owing to the potential higher leaky transcription of the gRNA-pMB1 specific to pTargetF in this strain. In this study, we modified the pCas/pTargetF system by replacing the promoter of gRNA-pMB1 with a tightly regulated promoter PrhaB, changing the replicon of pCas to a nontemperature-sensitive replicon, adding the sacB gene into pCas, and replacing the original N20-specific sequence of pTargetF with ccdB gene. We call this updated system as pEcCas/pEcgRNA. We found that gRNA-pMB1 indeed showed a slightly higher leaky expression in the pCas/pTargetF system compared with pEcCas/pEcgRNA. We also confirmed that genome editing can successfully be performed in BL21(DE3) by pEcCas/pEcgRNA with high efficiency. The application of pEcCas/pEcgRNA was then expanded to the E. coli B strain BL21 StarTM (DE3), K-12 strains MG1655, DH5α, CGMCC3705, Nissle1917, W strain ATCC9637, and also another species of Enterobacteriaceae, Tatumella citrea DSM13699, without any specific modifications. Finally, the plasmid curing process was optimized to shorten the time from $\sim$60 h to $\sim$32 h. The entire protocol (including plasmid construction, editing, electroporation and mutant verification, and plasmid elimination) took only $\sim$5.5 days per round in the pEcCas/pEcgRNA system, whereas it took $\sim$7.5 days in the pCas/pTargetF system. This study established a faster-acting genome editing tool that can be used in a wider range of E. coli strains and will also be useful for other Enterobacteriaceae species.
Topics: CRISPR-Cas Systems; Escherichia coli; Gene Editing; Genome, Bacterial; Plasmids
PubMed: 33764372
DOI: 10.1093/abbs/gmab036 -
Applied and Environmental Microbiology Apr 2015An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that...
An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea, another species of the Enterobacteriaceae, with highest efficiency of 100%.
Topics: CRISPR-Cas Systems; Enterobacteriaceae; Genes, Bacterial; Genetics, Microbial; Genome, Bacterial; Molecular Biology; Recombination, Genetic; Streptococcus pyogenes
PubMed: 25636838
DOI: 10.1128/AEM.04023-14 -
Frontiers in Microbiology 2022The genus (Diptera Tephritidae) includes some of the most important fruit fly pests in the Americas. Here, we studied the gut bacterial community of 3rd instar larvae...
The genus (Diptera Tephritidae) includes some of the most important fruit fly pests in the Americas. Here, we studied the gut bacterial community of 3rd instar larvae of sp. 1 through Next Generation Sequencing (lllumina) of the V3-V4 hypervariable region within the 16S rRNA gene. Gut bacterial communities were compared between host species (guava and peach), and geographical origins (Concordia and Horco Molle in Argentina) representing distinct ecological scenarios. In addition, we explored the effect of spatial scale by comparing the samples collected from different trees within each geographic origin and host species. We also addressed the effect of fruit size on bacterial diversity. The gut bacterial community was affected both by host species and geographic origin. At smaller spatial scales, the gut bacterial profile differed among trees of the same species and location at least in one host-location combination. There was no effect of fruit size on the larval gut bacteriome. Operational Taxonomic Units (OTUs) assigned to , and were identified in all samples examined, which suggest potential, non-transient symbioses. Better knowledge on the larval gut bacteriome contributes valuable information to develop sustainable control strategies against targeting key symbionts as the Achilles' heel to control this important fruit fly pest.
PubMed: 35359740
DOI: 10.3389/fmicb.2022.822990 -
Applied Microbiology and Biotechnology Mar 2021There is an increasing public awareness about the danger of dietary sugars with respect to their caloric contribution to the diet and the rise of overweight throughout...
There is an increasing public awareness about the danger of dietary sugars with respect to their caloric contribution to the diet and the rise of overweight throughout the world. Therefore, low-calorie sugar substitutes are of high interest to replace sugar in foods and beverages. A promising alternative to natural sugars and artificial sweeteners is the fructose derivative 5-keto-D-fructose (5-KF), which is produced by several Gluconobacter species. A prerequisite before 5-KF can be used as a sweetener is to test whether the compound is degradable by microorganisms and whether it is metabolized by the human microbiota. We identified different environmental bacteria (Tatumella morbirosei, Gluconobacter japonicus LMG 26773, Gluconobacter japonicus LMG 1281, and Clostridium pasteurianum) that were able to grow with 5-KF as a substrate. Furthermore, Gluconobacter oxydans 621H could use 5-KF as a carbon and energy source in the stationary growth phase. The enzymes involved in the utilization of 5-KF were heterologously overproduced in Escherichia coli, purified and characterized. The enzymes were referred to as 5-KF reductases and belong to three unrelated enzymatic classes with highly different amino acid sequences, activities, and structural properties. Furthermore, we could show that 15 members of the most common and abundant intestinal bacteria cannot degrade 5-KF, indicating that this sugar derivative is not a suitable growth substrate for prokaryotes in the human intestine. KEY POINTS: • Some environmental bacteria are able to use 5-KF as an energy and carbon source. • Four 5-KF reductases were identified, belonging to three different protein families. • Many gut bacteria cannot degrade 5-KF.
Topics: Bacteria; Clostridium; Fructose; Gammaproteobacteria; Gluconobacter; Humans; Sweetening Agents
PubMed: 33616697
DOI: 10.1007/s00253-021-11168-3 -
The Pan African Medical Journal 2021Tatumella ptyseos septicaemia in humans is yet to be reported in Nigeria with very few cases reported worldwide. This case report describes the clinical and distinctive...
Tatumella ptyseos septicaemia in humans is yet to be reported in Nigeria with very few cases reported worldwide. This case report describes the clinical and distinctive biochemical characteristics of Tatumella ptyseos, its antibiotic sensitivity pattern and risk factors associated with Tatumella ptyseos septicaemia. Our case is a 2 months old ex-premature female from Calabar, admitted in the month of May, 2018 into the Children´s Emergency Room, of the University of Calabar Teaching Hospital, Nigeria. She presented with cough of one month and fever of three weeks, and was found to be acutely ill looking, febrile with temperature of 38.6°C, mildly pale, dyspnoeic and tachypnoeic with SPO of 80% in room air, tender hepatomegaly of 6cm and a splenomegaly of 6cm. Blood culture yielded Gram negative rods identified as Tatumella ptyseos by OXOID MICROBACT™ GNB identification kit.
Topics: Bacteremia; Cough; Female; Fever; Gammaproteobacteria; Gram-Negative Bacterial Infections; Humans; Infant; Nigeria; Tertiary Care Centers
PubMed: 34178234
DOI: 10.11604/pamj.2021.39.6.25490 -
PloS One Mar 2011Necrotizing enterocolitis (NEC) affects 5-10% of infants born weighing less than 1500 g. Most models of NEC recapitulate late-stage disease with gut necrosis and...
INTRODUCTION
Necrotizing enterocolitis (NEC) affects 5-10% of infants born weighing less than 1500 g. Most models of NEC recapitulate late-stage disease with gut necrosis and elevated inflammatory mediators. Evaluation of NEC at earlier, less lethal stages of disease will allow investigation of initial disease triggers and may advance our understanding of temporal relationships between factors implicated in NEC pathogenesis. In this manuscript, we describe our investigation of early NEC and test the hypothesis that bacteria and inflammatory mediators differ between animals with early NEC and disease free animals.
METHODS
On DOL7 C3HeB/FeJ pups were fed liquid formula with 1×10(4) Streptococcus thoraltensis, Serratia marcescens, and Pseudomonas aeruginosa every 3 h. To initiate NEC, pups underwent asphyxia (100% N(2) for 90 s) and hypothermia (4°C for 10 min) after feeding. Pups were euthanized at 72 h. Intestines were collected for histologic NEC scoring and DNA/RNA extraction. Bacterial populations were identified by 16S rRNA pyrosequencing and principal component analysis (PCA). RNA isolates underwent QRT-PCR for Toll-like Receptor 4 (TLR4) and inducible nitric oxide synthase (iNOS).
RESULTS
Despite histologic, intestinal damage in mice with NEC, no gross necrosis was observed suggesting early disease. QRT-PCR yielded no difference between groups in TLR4 or iNOS mRNA levels. PCA demonstrated relative clustering of microbial communities based on presence or absence of NEC. 16S pyrosequencing demonstrated similar phyla between groups (Firmicutes and Proteobacteria predominated in all animals). However, the colonic microbiota of animals with NEC had more Citrobacter (p<0.01), Klebsiella (p<0.05), and Tatumella (p<0.05), while that of animals without NEC had more Streptococcus (p<0.01) and Enterococcus (p<0.01).
CONCLUSION
Citrobacter, Klebsiella, and Tatumella are associated with NEC. Differential colonic bacteria were identified despite the lack of inflammatory mediator elevation traditionally associated with NEC. This suggests a temporal relationship between bacteria and inflammatory mediators such that alterations in gut microbiota are associated with early NEC, while inflammatory mediator elevation is associated with advanced NEC.
Topics: Animals; Animals, Newborn; Base Sequence; DNA Primers; Enterocolitis, Necrotizing; Gram-Negative Bacteria; Mice; Mice, Inbred C3H; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 21445365
DOI: 10.1371/journal.pone.0018084 -
Molecules (Basel, Switzerland) Aug 2023Coffee fermentation is crucial for flavor and aroma, as microorganisms degrade mucilage and produce metabolites. This study aimed to provide a basis for understanding...
Coffee fermentation is crucial for flavor and aroma, as microorganisms degrade mucilage and produce metabolites. This study aimed to provide a basis for understanding the impact of microorganisms on from Yunnan, China, during washed processing. The microbial community structure and differentially changed metabolites (DCMs) of beans during washed processing were analyzed. The results indicated that the top five predominant microorganisms at the genera level were , , , , and for bacteria and , , , , and for fungi. Meanwhile, the relative content of 115 DCMs in 36 h samples decreased significantly, compared to non-fermentation coffee samples (VIP > 1, < 0.05, FC < 0.65), and the relative content of 28 DCMs increased significantly (VIP > 1, < 0.05, FC > 1.5). Furthermore, 17 DCMs showed a strong positive correlation with microorganisms, and 5 DCMs had a strong negative correlation ( < 0.05, |r| > 0.6). Therefore, the interaction and metabolic function of microbiota play a key role in the formation of coffee flavor, and these results help in clarifying the fermentation mechanisms of and in controlling and improving the quality of coffee flavor.
Topics: Coffea; Coffee; China; Fermentation; Microbiota; Saccharomycetales
PubMed: 37630344
DOI: 10.3390/molecules28166092 -
Frontiers in Microbiology 2018Bacteriologists have strived toward attaining a natural classification system based on evolutionary relationships for nearly 100 years. In the early twentieth century it...
Bacteriologists have strived toward attaining a natural classification system based on evolutionary relationships for nearly 100 years. In the early twentieth century it was accepted that a phylogeny-based system would be the most appropriate, but in the absence of molecular data, this approach proved exceedingly difficult. Subsequent technical advances and the increasing availability of genome sequencing have allowed for the generation of robust phylogenies at all taxonomic levels. In this study, we explored the possibility of linking biological characters to higher-level taxonomic groups in bacteria by making use of whole genome sequence information. For this purpose, we specifically targeted the genus and its four main lineages. The shared gene sets were determined for , the four lineages within the genus, as well as its sister-genus . This was followed by functional characterization of the gene sets using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In comparison to , various traits involved in nutrient cycling were identified within , providing evidence for increased efficacy in recycling of metabolites within the genus. Additionally, a number of traits associated with pathogenicity were identified within species often associated with opportunistic infections, with some support for adaptation toward overcoming host defenses. Some traits were also only conserved within specific lineages, potentially acquired in an ancestor to the lineage and subsequently maintained. It was also observed that the species isolated from the most diverse sources were generally the most versatile in their carbon metabolism. By investigating evolution, based on the more variable genomic regions, it may be possible to detect biologically relevant differences associated with the course of evolution and speciation.
PubMed: 29467735
DOI: 10.3389/fmicb.2018.00113 -
Scientific Reports Aug 2020Wine is a globally produced, marketed and consumed alcoholic beverage, which is valued for its aromatic and qualitative complexity and variation. These properties are...
Wine is a globally produced, marketed and consumed alcoholic beverage, which is valued for its aromatic and qualitative complexity and variation. These properties are partially attributable to the bacterial involvement in the fermentation process. However, the organizational principles and dynamic changes of the bacterial wine microbiota remain poorly understood, especially in the context of red and white wine variations and environmental stress factors. Here, we determined relative and absolute bacterial microbiota compositions from six distinct cultivars during the first week of fermentation by quantitative and qualitative 16S rRNA gene amplification and amplicon sequencing. All wines harboured complex and variable bacterial communities, with Tatumella as the most abundant genus across all batches, but red wines were characterized by higher bacterial diversity and increased relative and absolute abundance of lactic and acetic acid bacteria (LAB/AAB) and bacterial taxa of predicted environmental origin. Microbial diversity was positively correlated with plant-derived DNA concentrations in the wine and Botrytis cinerea infection before harvest. Our findings suggest that exogenous factors, such as procedural differences between red and white wine production and environmental stress on grape integrity, can increase bacterial diversity and specific bacterial taxa in wine, with potential consequences for wine quality and aroma.
Topics: Botrytis; Color; Crops, Agricultural; DNA, Plant; Food Quality; Gammaproteobacteria; Vitis; Wine
PubMed: 32796896
DOI: 10.1038/s41598-020-70535-8 -
Jundishapur Journal of Microbiology Jun 2014Tatumella ptyseos is a rod-shaped, Gram-negative, facultative, and anaerobic bacteria categorized in the Enterobacteriaceae family. It is a rare food-borne opportunistic...
Isolation, Identification and Antimicrobial Susceptibility Pattern of Tatumella ptyseos Strains Isolated From Powdered Infant Formula Milk Consumed in Neonatal Intensive Care Unit: First Report From Iran.
BACKGROUND
Tatumella ptyseos is a rod-shaped, Gram-negative, facultative, and anaerobic bacteria categorized in the Enterobacteriaceae family. It is a rare food-borne opportunistic pathogen which causes neonatal sepsis, bacteremia, and urinary tract infections. T. ptyseos has been also cultured from various food sources around the world.
OBJECTIVES
It is difficult to determine the source of the infection in the patients (especially newborns) due to low information about the epidemiology of T. ptyseos. The current study aimed to investigate the isolation, identification and antimicrobial susceptibility pattern of T. ptyseos strains from the consumed powdered infant formula milk (PIF) in hospital neonatal intensive care unit (NICU).
MATERIALS AND METHODS
A total of 125 powdered infant formula milk (PIF) samples were purchased from drug stores from June 2011 to March 2012. T. ptyseos was isolated according to food and drug administration (FDA) method. For final confirmation, biochemical testes embedded in API-20E system were used. Drug susceptibility test was performed using the disc diffusion method, according to clinical and laboratory standard institute (CLSI) recommendations.
RESULTS
Results of the study showed that, out of 125 samples, T. ptyseos was isolated from four (3/2%) PIF samples. All isolated strains (100%) were resistant to ampicillin, carbenicillin, cotrimoxazole and amoxicillin.
CONCLUSIONS
The present study was the first report on the isolation and identification of T. ptyseos from PIF in Iran. T. ptyseos are frequently present in various kinds of foods; therefore, further investigation on these samples is required. It is necessary to track the T. ptyseos in a wide variety of foods and individuals especially in immunocompromised people such as human immunodeficiency virus (HIV)-positive patients to reveal the possible routes of transmission of this pathogen to humans. In addition, molecular studies are required to determine the genetic relationship between T. ptyseos strains isolated from different sources.
PubMed: 25371802
DOI: 10.5812/jjm.10608