September 22, 2019  |  

Subaerial biofilms on granitic historic buildings: microbial diversity and development of phototrophic multi-species cultures.

Microbial communities of natural subaerial biofilms developed on granitic historic buildings of a World Heritage Site (Santiago de Compostela, NW Spain) were characterized and cultured in liquid BG11 medium. Environmental barcoding through next-generation sequencing (Pacific Biosciences) revealed that the biofilms were mainly composed of species of Chlorophyta (green algae) and Ascomycota (fungi) commonly associated with rock substrata. Richness and diversity were higher for the fungal than for the algal assemblages and fungi showed higher heterogeneity among samples. Cultures derived from natural biofilms showed the establishment of stable microbial communities mainly composed of Chlorophyta and Cyanobacteria. Although most taxa found in these cultures were not common in the original biofilms, they are likely common pioneer colonizers of building stone surfaces, including granite. Stable phototrophic multi-species cultures of known microbial diversity were thus obtained and their reliability to emulate natural colonization on granite should be confirmed in further experiments.

September 21, 2019  |  

Characterization of multi-drug resistant Enterococcus faecalis isolated from cephalic recording chambers in research macaques (Macaca spp.).

Nonhuman primates are commonly used for cognitive neuroscience research and often surgically implanted with cephalic recording chambers for electrophysiological recording. Aerobic bacterial cultures from 25 macaques identified 72 bacterial isolates, including 15 Enterococcus faecalis isolates. The E. faecalis isolates displayed multi-drug resistant phenotypes, with resistance to ciprofloxacin, enrofloxacin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, bacitracin, and erythromycin, as well as high-level aminoglycoside resistance. Multi-locus sequence typing showed that most belonged to two E. faecalis sequence types (ST): ST 4 and ST 55. The genomes of three representative isolates were sequenced to identify genes encoding antimicrobial resistances and other traits. Antimicrobial resistance genes identified included aac(6′)-aph(2″), aph(3′)-III, str, ant(6)-Ia, tetM, tetS, tetL, ermB, bcrABR, cat, and dfrG, and polymorphisms in parC (S80I) and gyrA (S83I) were observed. These isolates also harbored virulence factors including the cytolysin toxin genes in ST 4 isolates, as well as multiple biofilm-associated genes (esp, agg, ace, SrtA, gelE, ebpABC), hyaluronidases (hylA, hylB), and other survival genes (ElrA, tpx). Crystal violet biofilm assays confirmed that ST 4 isolates produced more biofilm than ST 55 isolates. The abundance of antimicrobial resistance and virulence factor genes in the ST 4 isolates likely relates to the loss of CRISPR-cas. This macaque colony represents a unique model for studying E. faecalis infection associated with indwelling devices, and provides an opportunity to understand the basis of persistence of this pathogen in a healthcare setting.

July 7, 2019  |  

Genome sequence of Pseudomonas parafulva CRS01-1, an antagonistic bacterium isolated from rice field.

Pseudomonas parafulva (formerly known as Pseudomonas fulva) is an antagonistic bacterium against several rice bacterial and fungal diseases. The total genome size of P. parafulva CRS01-1 is 5,087,619bp with 4389 coding sequences (CDSs), 77 tRNAs, and 7 rRNAs. The annotated full genome sequence of the P. parafulva CRS01-1 strain might shed light on its role as an antagonistic bacterium. Copyright © 2015. Published by Elsevier B.V.

July 7, 2019  |  

Whole-genome sequence of Serratia liquefaciens HUMV-21, a cytotoxic, auorum-sensing, and biofilm-producing clinical isolate.

A clinical isolate of Serratia liquefaciens (strain HUMV-21) was obtained from a skin ulcer of an adult patient. We report here its complete genome assembly using PacBio single-molecule real-time (SMRT) sequencing, which resulted in a single circular chromosome with 5.3 Mb. About 5,844 protein-coding genes are predicted from this assembly. Copyright © 2015 Lázaro-Díez et al.

July 7, 2019  |  

Complete genome sequence of Prevotella intermedia strain 17-2.

Prevotella intermedia, a Gram-negative black-pigmented anaerobic rod, is frequently isolated from not only periodontal pockets but also purulent infections. We report here the complete genome sequence of P. intermedia strain 17-2, which is a non-exopolysaccharide-producing variant obtained from exopolysaccharide (EPS)-producing P. intermedia strain 17 stock culture. Copyright © 2015 Nambu et al.

July 7, 2019  |  

Complete genome sequence of Marinobacter sp. CP1, isolated from a self-regenerating biocathode biofilm.

Marinobacter sp. CP1 was isolated from a self-regenerating and self-sustaining biocathode biofilm that can fix CO2 and generate electric current. We present the complete genome sequence of this strain, which consists of a circular 4.8-Mbp chromosome, to understand the mechanism of extracellular electron transfer in a microbial consortium. Copyright © 2015 Wang et al.

July 7, 2019  |  

Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens.

Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented ?imcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. These tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics. Copyright © 2015, Chan et al.

July 7, 2019  |  

Genome sequence of the Leisingera aquimarina type strain (DSM 24565(T)), a member of the marine Roseobacter clade rich in extrachromosomal elements.

Leisingera aquimarina Vandecandelaere et al. 2008 is a member of the genomically well characterized Roseobacter clade within the family Rhodobacteraceae. Representatives of the marine Roseobacter clade are metabolically versatile and involved in carbon fixation and biogeochemical processes. They form a physiologically heterogeneous group, found predominantly in coastal or polar waters, especially in symbiosis with algae, in microbial mats, in sediments or associated with invertebrates. Here we describe the features of L. aquimarina DSM 24565(T) together with the permanent-draft genome sequence and annotation. The 5,344,253 bp long genome consists of one chromosome and an unusually high number of seven extrachromosomal elements and contains 5,129 protein-coding and 89 RNA genes. It was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2010 and of the activities of the Transregional Collaborative Research Centre 51 funded by the German Research Foundation (DFG).

July 7, 2019  |  

Genome sequence of Phaeobacter caeruleus type strain (DSM 24564(T)), a surface-associated member of the marine Roseobacter clade.

In 2009 Phaeobacter caeruleus was described as a novel species affiliated with the marine Roseobacter clade, which, in turn, belongs to the class Alphaproteobacteria. The genus Phaeobacter is well known for members that produce various secondary metabolites. Here we report of putative quorum sensing systems, based on the finding of six N-acyl-homoserine lactone synthetases, and show that the blue color of P. caeruleus is probably due to the production of the secondary metabolite indigoidine. Therefore, P. caeruleus might have inhibitory effects on other bacteria. In this study the genome of the type strain DSM 24564(T) was sequenced, annotated and characterized. The 5,344,419 bp long genome with its seven plasmids contains 5,227 protein-coding genes (3,904 with a predicted function) and 108 RNA genes.

July 7, 2019  |  

Complete genome sequence of Lutibacter profundi LP1T isolated from an Arctic deep-sea hydrothermal vent system

Lutibacter profundi LP1T within the family Flavobacteriaceae was isolated from a biofilm growing on the surface of a black smoker chimney at the Loki’s Castle vent field, located on the Arctic Mid-Ocean Ridge. The complete genome of L. profundi LP1T is the first genome to be published within the genus Lutibacter. L. profundi LP1T consists of a single 2,966,978 bp circular chromosome with a GC content of 29.8%. The genome comprises 2,537 protein-coding genes, 40 tRNA species and 2 rRNA operons. The microaerophilic, organotrophic isolate contains genes for all central carbohydrate metabolic pathways. However, genes for the oxidative branch of the pentose-phosphate-pathway, the glyoxylate shunt of the tricarboxylic acid cycle and the ATP citrate lyase for reverse TCA are not present. L. profundi LP1T utilizes starch, sucrose and diverse proteinous carbon sources. In accordance, the genome harbours 130 proteases and 104 carbohydrate-active enzymes, indicating a specialization in degrading organic matter. Among a small arsenal of 24 glycosyl hydrolases, which offer the possibility to hydrolyse diverse poly- and oligosaccharides, a starch utilization cluster was identified. Furthermore, a variety of enzymes may be secreted via T9SS and contribute to the hydrolytic variety of the microorganism. Genes for gliding motility are present, which may enable the bacteria to move within the biofilm. A substantial number of genes encoding for extracellular polysaccharide synthesis pathways, curli fibres and attachment to surfaces could mediate adhesion in the biofilm and may contribute to the biofilm formation. In addition to aerobic respiration, the complete denitrification pathway and genes for sulphide oxidation e.g. sulphide:quinone reductase are present in the genome. sulphide:quinone reductase and denitrification may serve as detoxification systems allowing L. profundi LP1T to thrive in a sulphide and nitrate enriched environment. The information gained from the genome gives a greater insight in the functional role of L. profundi LP1T in the biofilm and its adaption strategy in an extreme environment.

July 7, 2019  |  

RelA mutant Enterococcus faecium with multiantibiotic tolerance arising in an immunocompromised host.

Serious bacterial infections in immunocompromised patients require highly effective antibacterial therapy for cure, and thus, this setting may reveal novel mechanisms by which bacteria circumvent antibiotics in the absence of immune pressure. Here, an infant with leukemia developed vancomycin-resistant Enterococcus faecium (VRE) bacteremia that persisted for 26 days despite appropriate antibiotic therapy. Sequencing of 22 consecutive VRE isolates identified the emergence of a single missense mutation (L152F) in relA, which constitutively activated the stringent response, resulting in elevated baseline levels of the alarmone guanosine tetraphosphate (ppGpp). Although the mutant remained susceptible to both linezolid and daptomycin in clinical MIC testing and during planktonic growth, it demonstrated tolerance to high doses of both antibiotics when growing in a biofilm. This biofilm-specific gain in resistance was reflected in the broad shift in transcript levels caused by the mutation. Only an experimental biofilm-targeting ClpP-activating antibiotic was able to kill the mutant strain in an established biofilm. The relA mutation was associated with a fitness trade-off, forming smaller and less-well-populated biofilms on biological surfaces. We conclude that clinically relevant relA mutations can emerge during prolonged VRE infection, causing baseline activation of the stringent response, subsequent antibiotic tolerance, and delayed eradication in an immunocompromised state.The increasing prevalence of antibiotic-resistant bacterial pathogens is a major challenge currently facing the medical community. Such pathogens are of particular importance in immunocompromised patients as these individuals may favor emergence of novel resistance determinants due to lack of innate immune defenses and intensive antibiotic exposure. During the course of chemotherapy, a patient developed prolonged bacteremia with vancomycin-resistant Enterococcus faecium that failed to clear despite multiple front-line antibiotics. The consecutive bloodstream isolates were sequenced, and a single missense mutation identified in the relA gene, the mediator of the stringent response. Strains harboring the mutation had elevated baseline levels of the alarmone and displayed heightened resistance to the bactericidal activity of multiple antibiotics, particularly in a biofilm. Using a new class of compounds that modulate ClpP activity, the biofilms were successfully eradicated. These data represent the first clinical emergence of mutations in the stringent response in vancomycin-resistant entereococci. Copyright © 2017 Honsa et al.

July 7, 2019  |  

Complete genome sequence of Staphylococcus epidermidis 1457.

Staphylococcus epidermidis 1457 is a frequently utilized strain that is amenable to genetic manipulation and has been widely used for biofilm-related research. We report here the whole-genome sequence of this strain, which encodes 2,277 protein-coding genes and 81 RNAs within its 2.4-Mb genome and plasmid. Copyright © 2017 Galac et al.

July 7, 2019  |  

Complete genome sequence of Citrobacter werkmanii strain BF-6 isolated from industrial putrefaction.

In our previous study, Citrobacter werkmanii BF-6 was isolated from an industrial spoilage sample and demonstrated an excellent ability to form biofilms, which could be affected by various environmental factors. However, the genome sequence of this organism has not been reported so far.We report the complete genome sequence of C. werkmanii BF-6 together with the description of the genome features and its annotation. The size of the complete chromosome is 4,929,789 bp with an average coverage of 137×. The chromosome exhibits an average G + C content of 52.0%, and encodes 4570 protein coding genes, 84 tRNA genes, 25 rRNA operons, 3 microsatellite sequences and 34 minisatellite sequences. A previously unknown circular plasmid designated as pCW001 was also found with a length of 212,549 bp and a G + C content of 48.2%. 73.5%, 75.6% and 92.6% of the protein coding genes could be assigned to GO Ontology, KEGG Pathway, and COG (Clusters of Orthologous Groups) categories respectively. C. werkmanii BF-6 and C. werkmanii NRBC 105721 exhibited the closest evolutionary relationships based on 16S ribosomal RNA and core-pan genome assay. Furthermore, C. werkmanii BF-6 exhibits typical bacterial biofilm formation and development. In the RT-PCR experiments, we found that a great number of biofilm related genes, such as bsmA, bssR, bssS, hmsP, tabA, csgA, csgB, csgC, csgD, csgE, and csgG, were involved in C. werkmanii BF-6 biofilm formation.This is the first complete genome of C. werkmanii. Our work highlights the potential genetic mechanisms involved in biofilm formation and paves a way for further application of C. werkmanii in biofilms research.

July 7, 2019  |  

Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum.

Ralstonia solanacearum is one of the most lethal phytopathogens in the world. Due to its broad host range, it can cause wilting disease in many plant species of economic interest. In this work, we identified the O-oligosaccharyltransferase (O-OTase) responsible for protein O-glycosylation in R. solanacearum. An analysis of the glycoproteome revealed that 20 proteins, including type IV pilins are substrates of this general glycosylation system. Although multiple glycan forms were identified, the majority of the glycopeptides were modified with a pentasaccharide composed of HexNAc-(Pen)-dHex3, similar to the O antigen subunit present in the lipopolysaccharide of multiple R. solanacearum strains. Disruption of the O-OTase led to the total loss of protein glycosylation, together with a defect in biofilm formation and reduced pathogenicity towards tomato plants. Comparative proteomic analysis revealed that the loss of glycosylation is not associated with widespread proteome changes. Only the levels of a single glycoprotein, the type IV pilin, were diminished in the absence of glycosylation. In parallel, disruption of glycosylation triggered an increase in the levels of a surface lectin homologous to Pseudomonas PA-IIL. These results reveal the important role of glycosylation in the pathogenesis of R. solanacearum. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail:

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