Ocean pollution is a worldwide environmental challenge that could be partially tackled through microbial applications. To shed light on the diversity and applications of the bacterial communities that inhabit the sediments trapped in artificial containers, we analyzed residues (Polyethylene terephthalate (PET) bottles and aluminum cans) collected from the Mediterranean Sea by scanning electron microscopy and Next Generation Sequencing. Moreover, we set a collection of culturable bacteria from the plastisphere that were screened for their ability to use PET as a carbon source. Our results reveal that Proteobacteria are the predominant phylum in all the samples and that Rhodobacteraceae, Woeseia, Actinomarinales, or Vibrio are also abundant in these residues. Moreover, we identified marine isolates with enhanced growth in the presence of PET: Aquimarina intermedia, Citricoccus spp., and Micrococcus spp. Our results suggest that the marine environment is a source of biotechnologically promising bacterial isolates that may use PET or PET additives as carbon sources.
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.
Although Streptococcus agalactiae periprosthetic joint infection (PJI) is not as prevalent as staphylococcal PJI, invasive S. agalactiae infection has recently increased in incidence. Here, RNA-Seq was used to perform transcriptomic analysis of S. agalactiae PJI using fluid derived from sonication of explanted arthroplasties of subjects with S. agalactiae PJI, with results compared to those of S. agalactiae strain NEM316 grown in vitro. 227 genes with outlier expression were found (164 up-regulated and 63 down-regulated) between PJI sonicate fluid and in vitro conditions. Functional enrichment analysis showed genes involved in mobilome and inorganic ion transport and metabolism to be most enriched. Genes involved in nickel, copper, and zinc transport, were upregulated. Among known virulence factors, cyl operon genes, encoding beta-hemolysin/cytolysin, were consistently highly expressed in PJI versus in vitro. The data presented provide insight into S. agalactiae PJI pathogenesis and may be a useful resource for the identification of novel PJI therapeutics or vaccines against invasive S. agalactiae infections.
Interspecific interactions within biofilms determine relative species abundance, growth dynamics, community resilience, and success or failure of invasion by an extraneous organism. However, deciphering interspecific interactions and assessing their contribution to biofilm properties and function remain a challenge. Here, we describe the constitution of a model biofilm composed of four bacterial species belonging to four different genera (Rhodocyclus sp., Pseudomonas fluorescens, Kocuria varians, and Bacillus cereus), derived from a biofilm isolated from an industrial milk pasteurization unit. We demonstrate that the growth dynamics and equilibrium composition of this biofilm are highly reproducible. Based on its equilibrium composition, we show that the establishment of this 4-species biofilm is highly robust against initial, transient perturbations but less so towards continuous perturbations. By comparing biofilms formed from different numbers and combinations of the constituent species and by fitting a growth model to the experimental data, we reveal a network of dynamic, positive, and negative interactions that determine the final composition of the biofilm. Furthermore, we reveal that the molecular determinant of one negative interaction is the thiocillin I synthesized by the B. cereus strain, and demonstrate its importance for species distribution and its impact on robustness by mutational analysis of the biofilm ecosystem.
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.
To understand antibiotic resistance in pathogenic bacteria, we need to monitor environmental microbes as reservoirs of antimicrobial resistance genes (ARGs). These bacteria are present in the air and can be investigated with the whole metagenome shotgun sequencing approach. This study aimed to investigate the feasibility of a method for metagenomic analysis of microbial composition and ARGs in the outdoor air. Air samples were collected with a Harvard impactor in the PM10 range at 50 m from a hospital in Budapest. From the DNA yielded from samples of PM10 fraction single-end reads were generated with an Ion Torrent sequencer. During the metagenomic analysis, reads were classified taxonomically. The core bacteriome was defined. Reads were assembled to contigs and the ARG content was analyzed. The dominant genera in the core bacteriome were Bacillus, Acinetobacter, Leclercia and Paenibacillus. Among the identified ARGs best hits were vanRA, Bla1, mphL, Escherichia coli EF-Tu mutants conferring resistance to Pulvomycin, BcI, FosB, and mphM. Despite the low DNA content of the samples of PM10 fraction, the number of detected airborne ARGs was surprisingly high.
Identification, source tracking, and surveillance of food pathogens is a crucial factor for the food-producing industry. Over the last decade, the techniques used for this have moved from conventional enrichment methods, through species-specific detection by PCR to sequencing-based methods, whole-genome sequencing (WGS) being the ultimate method. However, using WGS requires the right infrastructure, high computational power, and bioinformatics expertise. Therefore, there is a need for faster, more cost-effective, and more user-friendly methods. A newly developed method, ON-rep-seq, combines the classical rep-PCR method with nanopore sequencing, resulting in a highly discriminating set of sequences that can be used for species identification and also strain discrimination. This study is essentially a real industry case from a salmon processing plant. Twenty Listeria monocytogenes isolates were analyzed both by ON-rep-seq and WGS to identify and differentiate putative L. monocytogenes from a routine sampling of processing equipment and products, and finally, compare the strain-level discriminatory power of ON-rep-seq to different analyzing levels delivered from the WGS data. The analyses revealed that among the isolates tested there were three different strains. The isolates of the most frequently detected strain (n=15) were all detected in the problematic area in the processing plant. The strain level discrimination done by ON-rep-seq was in full accordance with the interpretation of WGS data. Our findings also demonstrate that ON-rep-seq may serve as a primary screening method alternative to WGS for identification and strain-level differentiation for surveillance of potential pathogens in a food-producing environment.
Background: Candida auris is an emerging fungal pathogen of worldwide interest. It is associated with high mortality rates and exhibits increased resistance to antifungals. Ultraviolet-C (UVC) light can be used to disinfect surfaces to mitigate its spread. In this study, we analyzed the performance of different UVC wavelengths against C. auris to determine its wavelength sensitivity and UVC dose requirements and evaluated biofilm prevention dose requirements on most used materials in healthcare settings. Objectives: 1. To investigate UVC disinfection performances and wavelength sensitivity of C. auris; 2. To evaluate the UVC dose required for prevention of biofilm prevention on stainless steel. Methods: C. auris was grown following standard procedures. The study utilized six different UVC LED arrays with wavelengths between 252 and 280 nm. Arrays were set at similar intensities, to obtain doses of 5-40 mJcm-2 and similar irradiation time. Disinfection performance for each array was determined using log reduction value (LRV) and percentage reduction by comparing the controls against the irradiated treatments. Evaluation of the ability of 267 nm UVC LEDs to prevent C. auris biofilm formation was investigated using stainless steel, plastic coupons, and poly-cotton fabric. Results: Peak sensitivity to UVC disinfection was between 267 and 270 nm. With 20 mJcm-2, the study obtained LRV 3. On steel coupons, 30 mJcm-2 was sufficient to prevent biofilm formation, on plastic only 10 mJcm-2. A dose of 60 mJcm-2 reduced biofilms on poly-cotton fabric significantly. Conclusions: Results revealed that C. auris was most susceptible at 267-270 nm. Additional highlights from the study allow for the design and implementation of disinfection systems.
Vibrio alginolyticus and Vibrio (Aliivibrio) fischeri are Gram-negative bacteria found globally in marine environments. During the past decade, studies have shown that certain Gram-negative bacteria, including Vibrio species (cholerae, parahaemolyticus, and vulnificus) are capable of using exogenous polyunsaturated fatty acids (PUFAs) to modify the phospholipids of their membrane. Moreover, exposure to exogenous PUFAs has been shown to affect certain phenotypes that are important factors of virulence. The purpose of this study was to investigate whether V. alginolyticus and V. fischeri are capable of responding to exogenous PUFAs by remodeling their membrane phospholipids and/or altering behaviors associated with virulence. Thin-layer chromatography (TLC) analyses and ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC/ESI-MS) confirmed incorporation of all PUFAs into membrane phosphatidylglycerol and phosphatidylethanolamine. Several growth phenotypes were identified when individual fatty acids were supplied in minimal media and as sole carbon sources. Interestingly, several PUFAs acids inhibited growth of V. fischeri. Significant alterations to membrane permeability were observed depending on fatty acid supplemented. Strikingly, arachidonic acid (20:4) reduced membrane permeability by approximately 35% in both V. alginolyticus and V. fischeri. Biofilm assays indicated that fatty acid influence was dependent on media composition and temperature. All fatty acids caused decreased swimming motility in V. alginolyticus, while only linoleic acid (18:2) significantly increased swimming motility in V. fischeri. In summary, exogenous fatty acids cause a variety of changes in V. alginolyticus and V. fischeri, thus adding these bacteria to a growing list of Gram-negatives that exhibit versatility in fatty acid utilization and highlighting the potential for environmental PUFAs to influence phenotypes associated with planktonic, beneficial, and pathogenic associations.
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.
The human microbiome has begun to emerge as a potential forensic tool, with varied applications ranging from unique identification to investigative leads that link individuals and/or locations. The relative abundance of the combined DNA of the microbiome, compared to human nuclear DNA, may expand potential sources of biological evidence, especially in cases with transfer or low-copy number DNA samples. This work sought to determine the optimal swab type for the collection and analysis of the microbiome. A bacterium (Proteus mirabilis) was deposited by pipette onto four swab types (cotton, flocked, dental applicators, and dissolvable), and extraction and real-time PCR quantitation of the bacterial DNA were performed, which allowed for absolute microbial DNA recovery and comparison of yields across the four sampling substrates. Flocked swabs had the highest yield (~1240 ng) compared to the cotton swabs (~184 ng), dental applicators (~533 ng), and dissolvable swabs (~430 ng). The collection efficiency was further evaluated for cotton and flocked swabs using dried microbial samples spotted onto non-porous surfaces (treated wood, glass, plastic, and tile). Flocked swabs performed consistently better across wood, glass, and tile, but showed decreased recovery from plastic. The cotton swabs failed in the recovery of P. mirabilis DNA across all surfaces. Knowing the appropriate sampling substrate will be useful as others continue to investigate the use of the microbiome as a forensics tool.
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.
M-protein (PAM) largely contributes to the pathogenesis of Pattern D Group A Streptococcus pyogenes (GAS). However, the mechanism of complex formation is unknown. In a system consisting of a Class II PAM from Pattern D GAS isolate NS88.2 (PAMNS88.2), with one K2hPg binding a-repeat in its A-domain, we employed biophysical techniques to analyze the mechanism of the K2hPg/PAMNS88.2 interaction. We show that apo-PAMNS88.2 is a coiled-coil homodimer (M.Wt. ~80 kDa) at 4°C - 25°C, and is monomeric (M.Wt. ~40 kDa) at 37°C, demonstrating a temperature-dependent dissociation of PAMNS88.2 over a narrow temperature range. PAMNS88.2 displayed a single tight binding site for K2hPg at 4°C, which progressively increased at 25°C through 37°C. We isolated the K2hPg/PAMNS88.2 complexes at 4°C, 25°C, and 37°C and found molecular weights of ~50 kDa at each temperature, corresponding to a 1:1 (m:m) K2hPg/PAMNS88.2 monomer complex. hPg activation experiments by streptokinase demonstrated that the hPg/PAMNS88.2 monomer complexes are fully functional. The data show that PAM dimers dissociate into functional monomers at physiological temperatures or when presented with the active hPg module (K2hPg) showing that PAM is a functional monomer at 37°C.
The transcription machinery of archaea can be roughly classified as a simplified version of eukaryotic organisms. The basal transcription factor machinery binds to the TATA-box found around 28 nucleotides upstream of the transcription start site; however, some transcription units lack a clear TATA-box and still have TBP/TFB binding over them. This apparent absence of conserved sequences could be a consequence of sequence divergence associated with the upstream region, operonic and gene organization. Furthermore, earlier studies have found that a structural analysis gains more information compared to a simple sequence inspection. In this work, we evaluated and coded 3630 archaeal promoter sequences of three organisms, Haloferax volcanii, Thermococcus kodakarensis, and Sulfolobus solfataricus into DNA duplex stability, enthalpy, curvature, and bendability parameters. We also split our dataset into conserved TATA and degenerated TATA promoters in order to identify differences among these two classes of promoters. The structural analysis reveals variations in archaeal promoters’ architecture, i.e., a distinctive signal is observed in the TFB, TBP, and TFE binding sites independently of these being TATA-conserved or TATA-degenerated. In addition, the promoter encountering method was validated with upstream regions of 13 other archaea, suggesting that there might be promoter sequences among them. Therefore, we suggest a novel method for locating promoters within the genome of archaea based on energetic/structural features.
The filamentous fungus Magnaporthe oryzae has the potential to be developed as an alternative platform organism for the heterologous production of industrially important enzymes. M. oryzae is easy to handle, fast-growing and unlike yeast, posttranslational modifications like N-glycosylations are similar to the human organism. Here, we established M. oryzae as a host for the expression of the unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO). UPOs are attractive biocatalysts for selective oxyfunctionalization of non-activated carbon-hydrogen bonds. To improve and simplify the isolation of AaeUPO in M. oryzae, we fused a Magnaporthe signal peptide for protein secretion and set it under control of the strong EF1-promotor. The success of the heterologous production of full-length AaeUPO in M. oryzae and the secretion of the functional enzyme was confirmed by a peroxygenase-specific enzyme assay. These results offer the possibility to establish the filamentous ascomycete M. oryzae as a broad applicable alternative expression system. This is in particular valid for proteins that cannot or not in sufficient yields produced in established systems.
Cyanobacterial carbonate precipitation induced by cells and extracellular polymeric substances (EPS) enhances mortar durability. The percentage of cell/EPS attachment regulates the effectiveness of the mortar restoration. This study investigates the cell coverage on mortar and microbially induced carbonate precipitation. Statistical analysis of results from scanning electron and fluorescence microscopy shows that the cell coverage was higher in the presence of UV-killed cells than living cells. Cells preferably attached to cement paste than sand grains, with a difference of one order of magnitude. The energy-dispersive X-ray spectroscopy analyses and Raman mapping suggest cyanobacteria used atmospheric CO2 to precipitate carbonates.
Microbially influenced corrosion (MIC) may contribute significantly to overall corrosion risks, especially in the gas and petroleum industries. In this study, we isolated four Prolixibacter strains, which belong to the phylum Bacteroidetes, and examined their nitrate-respiration- and Fe0-corroding activities, together with two previously isolated Prolixibacter strains. Four of the six Prolixibacter strains reduced nitrate under anaerobic conditions, while the other two strains did not. The anaerobic growth of the four nitrate-reducing strains was enhanced by nitrate, which was not observed in the two nitrate-non-reducing strains. When the nitrate-reducing strains were grown anaerobically in the presence of Fe0 or carbon steel, the corrosion of the materials was enhanced by more than 20-fold compared to that in aseptic controls. This enhancement was not observed in cultures of the nitrate-non-reducing strains. The oxidation of Fe0 in the anaerobic cultures of nitrate-reducing strains occurred concomitantly with the reduction of nitrite. Since nitrite chemically oxidized Fe0 under anaerobic and aseptic conditions, the corrosion of Fe0- and carbon-steel by the nitrate-reducing Prolixibacter strains was deduced to be mainly enhanced via the biological reduction of nitrate to nitrite, followed by the chemical oxidation of Fe0 to Fe2+ and Fe3+ coupled to the reduction of nitrite.
Denitrification plays a central role in the global nitrogen cycle, reducing and removing nitrogen from marine and terrestrial ecosystems. The flux of nitrogen species through this pathway has a widespread impact, affecting ecological carrying capacity, agriculture, and climate. Nitrite reductase (Nir) and nitric oxide reductase (NOR) are the two central enzymes in this pathway. Here we present a previously unreported Nir domain architecture in members of Phylum Chloroflexi. Phylogenetic analyses of protein domains within Nir indicate that an ancestral horizontal transfer and fusion event produced this chimeric domain architecture. We also identify an expanded genomic diversity of a rarely reported nitric oxide reductase subtype, eNOR. Together, these results suggest a greater diversity of denitrification enzyme arrangements exist than have been previously reported.