Carotenoids are widely used in functional foods, cosmetics, and health supplements, and their importance and scope of use are continuously expanding. Here, we characterised carotenoid biosynthetic genes of the plant-pathogenic bacterium Pantoea ananatis, which carries a carotenoid biosynthetic gene cluster (including crtE, X, Y, I, B, and Z) on a plasmid. Reverse transcription–polymerase chain reaction (RT-PCR) analysis revealed that the crtEXYIB gene cluster is transcribed as a single transcript and crtZ is independently transcribed in the opposite direction. Using splicing by overlap extension with polymerase chain reaction (SOE by PCR) based on asymmetric amplification, we reassembled crtE–B, crtE–B–I, and crtE–B–I–Y. High-performance liquid chromatography confirmed that Escherichia coli expressing the reassembled crtE–B, crtE–B–I, and crtE–B–I–Y operons produced phytoene, lycopene, and β-carotene, respectively. We found that the carotenoids conferred tolerance to UV radiation and toxoflavin. Pantoea ananatis shares rice environments with the toxoflavin producer Burkholderia glumae and is considered to be the first reported example of producing and using carotenoids to withstand toxoflavin. We confirmed that the carotenoid production of P. ananatis is dependent on RpoS, which is positively regulated by Hfq/ArcZ and negatively regulated by ClpP, similar to an important regulatory network of E. coli (HfqArcZ → RpoS Ͱ ClpXP). We also demonstrated that Hfq-controlled quorum signalling de-represses EanR to activate RpoS, thereby initiating carotenoid production. Survival genes such as those responsible for the production of carotenoids of the plant-pathogenic P. ananatis must be expressed in a timely manner to overcome stressful environments and compete with other microorganisms. This mechanism is likely maintained by a brake with excellent performance, such as EanR.

Actinobacteria are important cave inhabitants, but knowledge of how anthropization and anthropization-related visual marks affect this community on cave walls is lacking. We compared Actinobacteria communities among four French limestone caves (Mouflon, Reille, Rouffignac, Lascaux) ranging from pristine to anthropized, and within Lascaux Cave between marked (wall visual marks) and unmarked areas in different rooms (Sas-1, Passage, Apse, Diaclase). In addition to the 16S rRNA gene marker, 441 bp fragments of the hsp65 gene were used and an hsp65-related taxonomic database was constructed for identification of Actinobacteria to the species level by Illumina-MiSeq analysis. The hsp65 marker revealed higher resolution for species and higher richness (99% OTU cutoff) versus 16S rRNA gene; however, more taxa were identified at higher taxonomic ranks. Actinobacteria communities varied between Mouflon and Reille caves (both pristine), and Rouffignac and Lascaux (both anthropized). Rouffignac displayed high diversity of Nocardia, suggesting human inputs, and Lascaux exhibited high Mycobacterium relative abundance, whereas Gaiellales were typical in pristine caves and the Diaclase (least affected area of Lascaux Cave). Within Lascaux, Pseudonocardiaceae dominated on unmarked walls and Streptomycetaceae (especially Streptomyces mirabilis) on marked walls, indicating a possible role in mark formation. A new taxonomic database (https://zenodo.org/record/5576074) was developed. Although not all Actinobacteria species were represented, the use of the hsp65 marker enabled species-level variations of the Actinobacteria community to be documented based on the extent of anthropogenic pressure. This approach proved effective when comparing different limestone caves or specific conditions within one cave.

To survive within complex environmental niches, including the human host, bacteria have evolved intricate inter-species communities driven by competition for limited nutrients, cooperation via complementary metabolic proficiencies, and establishment of homeostatic relationships with the host immune system. Such complex, interdependent relationships have hampered attempts to culture many bacterial strains in research settings, where standard readout of co-culture experiments are usually limited to the relative abundance of each species. Here, we utilize a microfluidic-based co-culture system to characterize dynamic interactions between multiple oral bacterial isolates. Using time-lapse imaging, we define species-specific effects on spatial community relationships during co-culture of Streptococcus species and Staphylococcus aureus with Actinomyces species. Co-culture of Streptococcus cristatus or S. salivarius in nanoliter compartments with Actinomyces graevenitzii revealed localized exclusion of Streptococcus and Staphylococcus from media immediately surrounding A. graevenitzii micro colonies. This community structure did not occur with S. mitis or S. oralis strains, or in co-cultures containing other Actinomycetaceae species such as S. odontolyticus or A. naeslundii. Moreover, fewer neutrophils were attracted to compartments containing both A. graevenitzii and Staphylococcus aureus than to equal number of either species alone, suggesting a possible survival benefit from the interaction.

Laccases belong to a family of multicopper enzymes able to oxidize a broad spectrum of organic compounds. Despite the well-known property of laccases to carry out bleaching and degradation of industrial dyes and polyphenolic compounds, their industrial use is often limited by the high cost, low efficiency, or instability of these enzymes. To look for new microorganisms which produce laccases that are potentially suitable for industrial applications, we have isolated several fungal strains from a cave in northern Spain. Their phenotypic analysis on agar plates supplemented with ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) disclosed two laccase-positive strains. Further genotyping revealed that they belonged to the Gliomastix murorum and Conidiobolus thromboides species. The secretion of G. murorum and C. thromboides laccase-like enzymes was then confirmed by zymography. Further identification of these polypeptides by mass-spectroscopy revealed the nature of the laccases and made it possible to predict their functional domains and other features. In addition, plate assays revealed that the laccases secreted by both G. murorum and C. thromboides were capable of degrading industrial dyes (Congo Red, Indigo, and Eriochrome Black T). Homology modeling and substrate docking predicted the putative structure of the currently uncrystallized G. murorum enzyme as well as its amino acid residues potentially involved in interactions with these dyes. In summary, new biochemical and structural insights into decolorization mediated by G. murorum laccase as well as identification of laccase-like oxidase in C. thromboides point to a promising future for these enzymes in biotechnology.

In fungal pathogens, the transcription factor SrbA (a sterol regulatory element-binding protein, SREBP) and CBC (CCAAT binding complex) have been reported to regulate azole resistance by competitively binding the TR34 region (34 mer) in the promoter of the drug target gene, erg11A. However, current knowledge about how the SrbA and CBC coordinately mediate erg11A expression remains limited. In this study, we uncovered a novel relationship between HapB (a subunit of CBC) and SrbA in which deletion of hapB significantly prolongs the nuclear retention of SrbA by increasing its expression and cleavage under azole treatment conditions, thereby enhancing Erg11A expression for drug resistance. Furthermore, we verified that loss of HapB significantly induces the expression of the rhomboid protease RbdB, Dsc ubiquitin E3 ligase complex, and signal peptide peptidase SppA, which are required for the cleavage of SrbA, suggesting that HapB acts as a repressor for these genes which contribute to the activation of SrbA by proteolytic cleavage. Together, our study reveals that CBC functions not only to compete with SrbA for binding to erg11A promoter region but also to affect SrbA expression, cleavage, and translocation to nuclei for the function, which ultimately regulate Erg11A expression and azole resistance.

Saccharomyces cerevisiae produces a multicellular phenotype, known as a mat, on a semi-solid medium. This biofilm phenotype was first described in the lab strain 1278b and has been analyzed mostly in this same background. Yeast cells form a mat by spreading across the medium and adhering to each other and the surface, in part through the variegated expression of the cell adhesin, FLO11. This process creates a characteristic floral pattern and generates pH and glucose gradients outward from the center of the mat. Mats are encapsulated in a liquid which may aid in surface spreading and diffusion. Here, we examine thirteen environmental isolates that vary visually in the phenotype. We predicted that mat properties were universal and increased morphological complexity would be associated with more extreme trait values. Our results showed that pH varied significantly among strains, but was not correlated to mat complexity. Only two isolates generated significant liquid boundaries and neither produced visually complex mats. In five isolates, we tracked the initiation of FLO11 using GFP under the control of the endogenous promoter. Strains varied in when and how much GFP was detected, with increased signal associated with increased morphological complexity. Generally, the signal was strongest in the center of the mat and absent at the expanding edge. Our results show that traits discovered in one background vary and exist independently of mat complexity in natural isolates. The environment may favor different sets of traits, which could have implications for how this yeast adapts to its many ecological niches.

The ability of S. aureus to infect bone and osteoblasts is correlated to its incredible virulence armamentarium that can mediate the invasion/internalization process, cytotoxicity, membrane damage and intracellular persistence. We comparatively analyzed the interaction, persistence and modulation of expression of selected genes as well as cell viability in an ex-vivo model using human MG-63 osteoblasts of two previously studied and well-characterized S. aureus clinical strains belonging to ST239-SCCmecIII-t037 and ST228-SCCmecI-t041 clones at 3h and 24h post-infection (p.i). ATCC12598 was used as a control strain. Using Imaging Flow Cytometry analysis, we found that strains differently invaded osteoblasts after 3h and 24h: ATCC12598 internalized in 70% and 50% of cells, ST239-SCCmecIII in 50% and 45% and ST228-SCCmecI in 30% and 20%, respectively. ST239-III, during the infection period, exerted a significative cytotoxic activity due to the over-expression of hla and psmA and the increased expression of the genes involved in adhesion, probably due to the release and re-entry of bacteria inside MG-63 at 24h p.i. The lower invasiveness of ST228-I was also correlated with the non-cytotoxic activity inside osteoblasts. This clone was not able to activate a sufficient cellular reaction and succumbed in-side the MG-63 cells.

The goal of this study was to design genus-specific primers for rapid evaluation of the most abundant bacterial genera identified using amplicon-based sequencing of the 16S rRNA gene in fish-related samples and surrounding water. Efficient genus-specific primers were designed for eleven bacterial genera including Alkalimarinus, Colwellia, Enterovibrio, Marinomonas, Massilia, Oleispira, Phaeobacter, Photobacterium, Polarbacerium, Pseudomonas, and Psychrobium. The specificity of the primers was confirmed by the phylogeny of the sequenced polymerase chain reaction (PCR) amplicons that indicated primers were genus-specific except in the case of Colwellia and Phaeobacter. Copy number of the 16S rRNA gene obtained by quantitative PCR using genus-specific primers and the relative abundance obtained by 16S rRNA gene sequencing using universal primers were well correlated for the five analyzed abundant bacterial genera. Low correlations between quantitative PCR and 16S rRNA gene sequencing for Pseudomonas were explained by the higher coverage of known Pseudomonas species by the designed genus-specific primers than the universal primers used in 16S rRNA gene sequencing. The designed genus-specific primers are proposed as rapid and cost-effective tools to evaluate the most abundant bacterial genera in fish-related or potentially other metagenomics samples.

We report here the complete genome sequence of the Rhizobium rhizogenes (formerly Agrobacterium rhizogenes) strain LBA9402 (NCPPB1855rifR), a pathogenic strain causing hairy root disease. In order to assemble a complete genome we obtained short-reads from Illumina sequencing as well as long-reads from Oxford Nanopore Technology sequencing. The genome consists of a 3,958,212 bp chromosome, a 2,005,144 bp chromid (secondary chromosome) and a 252,168 bp Ri plasmid (pRi1855), respectively. The primary chromosome was very similar to that of the avirulent biocontrol strain K84, but the chromid showed a 724 kbp deletion accompanied by a large 1.8 Mbp inversion revealing the dynamic nature of these secondary chromosomes. The sequence of the agropine Ri plasmid was compared to other types of Ri and Ti plasmids. Thus we identified the genes responsible for agropine catabolism, but also a unique segment adjacent to the TL-region that has the signature of a new opine catabolic gene cluster including the three genes that together encode an opine dehydrogenase. Our sequence analysis also revealed a novel gene at the very right end of the TL-DNA, which is unique for the agropine Ri plasmid. The protein encoded by this gene was most related to the succinamopine synthases of chrysopine and agropine Ti plasmids and thus may be involved in synthesis of the unknown opine that can be degraded by the adjacent catabolic cluster. The available sequence will facilitate the use of R. rhizogenes and especially LBA9402 in both the laboratory and for biotechnological purposes.

Temperate phages are bacterial viruses that either reside integrated in a bacterial genome as lysogens or enter a lytic lifecycle. Decision between lifestyles is determined by a switch involving a phage-encoded repressor, CI, and a promoter region from which lytic and lysogenic genes are divergently transcribed. Here we investigate the switch of phage phi13 from the human pathogen Staphylococcus aureus. phi13 encodes several virulence factors and is prevalent in S. aureus strains colonizing humans. We show that the phi13 switch harbors a cI gene, a predicted mor (modulator of repression) gene, and three high-affinity operator sites binding CI. To quantify the decision between lytic and lysogenic lifestyle, we introduced reporter plasmids that carry the 1.3 kb switch region from phi13 with the lytic promoter fused to lacZ into S. aureus and B. subtilis. Analysis of beta-galactosidase expression indicated that decision frequency is independent of host factors. The white “lysogenic” phenotype, which relies on expression of cI, could be switched to a stable blue “lytic” phenotype by DNA damaging agents. We have characterized lifestyle decisions of phage phi13, and our approach may be applied to other temperate phages encoding virulence factors in S. aureus.

The basal zone of glaciers is characterised by physicochemical properties that are distinct from firnified ice because of strong interactions with underlying substrate. Basal ice ecology and the roles that the microbiota play in biogeochemical cycling, weathering, and proglacial soil formation, remains poorly known. We report bacterial diversity and potential ecological roles at three temperate Icelandic glaciers. We sampled three physically distinct basal ice facies (stratified, dispersed, debris bands) and found biological similarities and differences between them; basal ice character is therefore an important sampling consideration in future studies. High abundance of silicates and Fe-containing minerals could sustain the basal ice ecosystem, in which chemolithotrophic bacteria (~23%), especially Fe-oxidisers and hydrogenotrophs, can fix C, which can be utilised by heterotrophs. Methanogenic-affiliated detected sequences showed that silicate comminution-derived hydrogen can also be utilised for methanogenesis. Metabolism predicted by 16S rRNA diversity revealed that methane metabolism and C-fixation are the most common pathways, indicating the importance of these metabolic routes. Carbon concentrations were low compared to other ecosystems, but we report the highest carbon concentration in basal ice to date. Carbon release from melting basal ice may play an important role in promoting pioneering communities establishment and soil development in deglaciating forelands.

Listeria monocytogenes is a human pathogen. It is the causative agent of listeriosis, the leading cause of bacterial-linked foodborne mortality in Europe and elsewhere. Outbreaks of listeriosis have been associated with the consumption of fresh produce including vegetables and fruits. In this review we summarize current data providing direct or indirect evidence that plants can serve as habitat for L. monocytogenes, enabling this human pathogen to survive and grow. The current knowledge of the mechanisms involved in the interaction of this bacterium with plants is addressed, and whether this foodborne pathogen elicits an immune response in plants is discussed.

In the microbiological diagnosis of bloodstream infections (BSI), blood culture (BC) is considered the gold standard test despite its limitations such as low sensitivity and slow turnaround time. A new FDA-cleared and CE-marked platform utilizing magnetic resonance to detect amplified DNA of the six most common and/or problematic BSI pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli) is available and may shorten the time to diagnosis and potentially improve antimicrobial utilization. Whole blood samples from hospitalized patients with clinical signs of sepsis were analyzed using the T2Bacteria Panel (T2Biosystems) and compared to simultaneously collected BC. Discrepant results were evaluated based on clinical infection criteria, combining supporting culture results and the opinion of treating physicians. A total of 55 samples from 53 patients were evaluated. The sensitivity and specificity of the T2Bacteria panel was 94% (16 out of 17 detections of T2Bacteria-targeted organisms) and 100%, respectively, with 36.4% (8 of 22) causes of BSI detected only by this method. The T2Bacteria Panel detected pathogens on average 55 hours faster than standard BC. In our study, 9 of 15 patients with positive T2Bacteria Panel results received early-targeted antibiotic therapy, and/or their antimicrobial treatment was modified based on T2Bacteria Panel findings. Given the high reliability, faster time to detection, and easy workflow, the technique qualifies as a point of care testing approach.

Antibacterial properties of toothpastes enable chemical plaque control in limited-access tooth regions that are mechanically not sufficiently reached by toothbrushes. Therefore, this study aimed to compare different microbial methods to assess antimicrobial toothpaste properties and evaluate different toothpastes in terms of their antibacterial efficacy against different oral microorganisms in vitro. Six toothpaste suspensions with varying antibacterial supplements were applied to a multi-species biofilm model (Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans) as well as to each microorganism. A culture method was used to assess the anti-biofilm effects and two different agar diffusion assays were performed for testing the antimicrobial effect on each microorganism. The measurements of the culture and diffusion analyses were statistically normalized and compared and toothpastes were ranked according to their antimicrobial efficacy. The results of both agar diffusion assays showed a high correlation across all tested species (Spearman correlation coefficients ρs > 0.95). The results of the multi-species biofilm model, however, substantially differed in its assessment of antibacterial properties (ρs ranging from 0.22 to 0.87), compared to the results of both diffusion assays. Toothpastes with amine fluoride with and without stannous fluoride, and triclosan resulted in the highest antimicrobial efficacy, while activated carbon supplements were comparable to the negative control NaCl. The appropriate selection of a broad range of oral microorganisms seems crucial when testing the chemical impact of toothpastes and toothpaste supplements.

DNA extraction and preservation bias is a recurring topic in DNA sequencing-based microbial ecology. Different methodologies can lead to distinct outcomes, which has been demonstrated especially in studies investigating prokaryotic community composition. Eukaryotic microbes are ubiquitous, diverse, and increasingly a subject of investigation in addition to bacteria and archaea. However, little is known about how the choice of DNA preservation and extraction methodology impacts perceived eukaryotic community composition. In this study, we compared the effect of two DNA preservation protocols and 6 DNA extraction methods on the community profiles of both eukaryotes and prokaryotes in phototrophic biofilms on seagrass (Zostera marina) leaves from the Baltic Sea. We found that, whereas DNA preservation and extraction method caused significant bias in perceived community composition for both eukaryotes and prokaryotes, extraction bias was more pronounced for eukaryotes than prokaryotes. Especially soft-bodied or hard-shelled eukaryotes like nematodes and diatoms were differentially abundant depending on the extraction method. We conclude that careful consideration of DNA preservation and extraction methodology is crucial to achieving representative community profiles of eukaryotes in marine biofilms, and likely all other habitats containing diverse eukaryotic microbial communities.

The dental clinic air microbiome incorporates microbes from the oral cavity and upper respiratory tract (URT). This study aimed to establish a reliable methodology for air sampling in a dental clinic setting and quantify the abundance of culturable mesophilic aerobic bacteria present in these samples using regression modeling. Staphylococcus hominis, a potentially pathogenic bacterium typically found in the human oropharynx and URT, was consistently isolated. S. hominis was the most abundant species of aerobic bacteria (22% to 24%) and comprised 60% to 80% of all Staphylococcus spp. The study also assessed the susceptibility of S. hominis to 222nm-far-UVC light in laboratory experiments, which showed an exponential surface inactivation constant of k = 0.475 cm2/mJ. This constant is a critical parameter for future on-site use of far-UVC light as a technique for reducing pathogenic bacterial load in dental clinics.

Given the increasing eutrophication of water bodies in Africa due to increasing anthropogenic pressures, data are needed to better understand the responses of phytoplankton communities to these changes in tropical lakes. These ecosystems are used by local human populations for multiple purposes, including fish and drinking water production, potentially exposing these populations to health threats if, for example, an increase in toxic cyanobacterial blooms is associated with increasing eutrophication. To test the short-term response of the phytoplankton community to the addition of nutrients (phosphorus and nitrogen, alone or in combination) and Nile tilapia, we developed an in situ mesocosm experiment in a freshwater lagoon located near Abidjan (Ivory Coast). We found that phytoplankton growth (estimated by chlorophyll-a quantification) was highly stimulated when both nitrogen and phosphorus were added, while there was no clear evidence for such colimitation by these two nutrients when considering their concentrations in the lagoon. Phytoplankton growth was accompanied by significant changes in the diversity and composition of this community and did not lead to an increase in the proportions of cyanobacteria. However, the addition of fish to some mesocosms resulted in a drastic decrease in phytoplankton biomass and a dominance of chlorophytes in this community. Finally, these experiments showed that the addition of nitrogen, alone or combined with phosphorus, stimulated microcystin production by cyanobacteria. In addition, no evidence for microcystin accumulation in the fish was found. Taken together, these data allow us to discuss strategies for controlling cyanobacterial blooms in this tropical ecosystem.

Bacillus subtilis is described as a promising production strain for lipopeptides. In the case of B. subtilis strains JABs24 and DSM10T, surfactin, and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho-transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In B. subtilis 168, a point mutation in the degQ promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14-fold reduced expression. This effect on degQ expression and the associated impact on lipopeptide formation was examined for B. subtilis JABs24, a lipopeptide-producing derivative of strain 168, and B. subtilis wild-type strain DSM10T, which has a native degQ expression. Based on the stimulatory effects of the DegU regulator on secretory protease formation, the impact of degQ expression on extracellular protease activity was additionally investigated. To follow the impact of degQ, a deletion mutant was constructed for DSM10T, while a natively expressed degQ version was integrated into strain JABs24. This allowed strain-specific quantification of the stimulatory effect of degQ expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10T. While an unaffected degQ expression reduced surfactin production in JABs24 by about 25%, a 6-fold increase in plipastatin was observed. In contrast, degQ deletion in DSM10T increased surfactin titer by 3-fold but decreased plipastatin production by 5-fold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.