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Paper mills processing recycled paper suffer from biofouling causing problems both in the mill and final product. The total bacterial community composition and identification of specific taxa in the process water and biofilms at the stock preparation and paper machine areas in a mill with recycled paper pulp was described by using a DNA-based approach. Process water in a similar mill was also analyzed to investigate if general trends can be found between mills and over time. Bacterial community profiles, analyzed by terminal-restriction fragment length polymorphism (T-RFLP), in process water showed that the dominant peaks in the profiles were similar between the two mills, although the overall composition was unique for each mill. When comparing process water and biofilm at different locations within one of the mills, we observed a separation according to location and sample type, with the biofilm from the paper machine being most different. 16S rRNA gene clone libraries were generated and 404 clones were screened by RFLP analysis. Grouping of RFLP patterns confirmed that the biofilm from the paper machine was most different. A total of 99 clones representing all RFLP patterns were analyzed, resulting in sequences recovered from nine bacterial phyla, including two candidate phyla. Bacteroidetes represented 45% and Actinobacteria 23% of all the clones. Sequences with similarity to organisms implicated in biofouling, like Chryseobacterium spp. and Brevundimonas spp., were recovered from all samples even though the mill had no process problems during sampling, suggesting that they are part of the natural paper mill community. Moreover, many sequences showed little homology to as yet uncultivated bacteria implying that paper mills are interesting for isolation of new organisms, as well as for bioprospecting.

India has the highest burden of Tuberculosis (TB) and multidrug-resistant TB (MDR-TB) worldwide. Innovative technology is the need of the hour to identify these cases that remain either undiagnosed or inadequately diagnosed due to the unavailability of appropriate tools at primary healthcare settings. We developed and evaluated 3 kits, namely ‘TB Detect’ (containing BioFM-Filter device), ‘TB Concentration and Transport’ (containing Trans-Filter device) and ‘TB DNA Extraction’ kits. These kits enable bio-safe equipment-free concentration of sputum on filters and improved fluorescence microscopy at primary healthcare centres, ambient temperature transport of dried inactivated sputum filters to central laboratories and molecular detection of drug resistance by PCR and DNA sequencing (Mol-DST). In a 2-site evaluation (n = 1190 sputum specimens) on presumptive TB patients, BioFM-Filter smear exhibited a significant increase in positivity of 7% and 4% over ZN smear and LED-FM smear (p

Most filamentous bacteria in biological nutrient removal (BNR) processes have not been identified beyond their morphotype and simple staining reactions. Furthermore, the majority of sludge filaments observed under the microscope do not hybridize to commonly used phylogenetic probes for well characterized bacterial phyla such as the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Specific 16S rRNA-targeted oligonucleotide probes were designed for the phylum Chloroflexi (green non-sulfur bacteria) and optimized for use in fluorescence in situ hybridization. Chloroflexi have been implicated in BNR systems by phylogenetic identification of filamentous bacteria isolated by micromanipulation from sludge and culture-independent molecular phylogenetic surveys. The predominant morphotype responding to the probes was filamentous and these filaments were generally abundant in 10 Australian full-scale and two laboratory-scale BNR samples examined. Filamentous bacteria responding to a subdivision 1 Chloroflexi probe were rare in the samples, whereas subdivision 3 Chloroflexi filaments were very common in some sludges. This is in direct contrast to results obtained from molecular phylogenetic surveys of BNR systems where most sludge 16S rDNA clones belong to subdivision 1 and only a few to subdivision 3. It is suggested that filamentous bacteria belonging to the Chloroflexi phylum account for a large fraction of phylogenetically uncharacterized filaments in BNR systems and are likely to be abundant in such systems on a global scale.

This special issue provides a selection of papers that cover the whole range of topics presented at ISBA16.

A total of 11 bacterial strains capable of completely degrading 2-butoxyethanol (2-BE) were isolated from forest soil, a biotrickling filter, a bioscrubber, and activated sludge, and identified by 16S rRNA gene sequence analysis. Eight of these strains belong to the genus Pseudomonas; the remaining three strains are Hydrogenophaga pseudoflava BOE3, Gordonia terrae BOE5, and Cupriavidus oxalaticus BOE300. In addition to 2-BE, all isolated strains were able to grow on 2-ethoxyethanol and 2-propoxyethanol, ethanol, n-hexanol, ethyl acetate, 2-butoxyacetic acid (2-BAA), glyoxylic acid, and n-butanol. Apart from the only gram-positive strain isolated, BOE5, none of the strains were able to grow on the nonpolar ethers diethyl ether, di-n-butyl ether, n-butyl vinyl ether, and dibenzyl ether, as well as on 1-butoxy-2-propanol. Strains H. pseudoflava BOE3 and two of the isolated pseudomonads, Pseudomonas putida BOE100 and P. vancouverensis BOE200, were studied in more detail. The maximum growth rates of strains BOE3, BOE100, and BOE200 at 30 °C were 0.204 h-1 at 4 mM, 0.645 h-1 at 5 mM, and 0.395 h-1 at 6 mM 2-BE, respectively. 2-BAA, n-butanol, and butanoic acid were detected as potential metabolites during the degradation of 2-BE. These findings indicate that the degradation of 2-BE by the isolated gram-negative strains proceeds via oxidation to 2-BAA with subsequent cleavage of the ether bond yielding glyoxylate and n-butanol. Since Gordonia terrae BOE5 was the only strain able to degrade nonpolar ethers like diethyl ether, the degradation pathway of 2-BE may be different for this strain., Projekt DEAL, Landesgraduiertenförderung der Universität Stuttgart

Activating industrially important aromatic hydrocarbons by installing halogen atoms is extremely important in organic synthesis and often improves the pharmacological properties of drug molecules. To this end, tryptophan halogenase enzymes are potentially valuable tools for regioselective halogenation of arenes, including various industrially important indole derivatives and similar scaffolds. Although endogenous enzymes show reasonable substrate scope towards indole compounds, their efficacy can often be improved by engineering. Using a structure‐guided semi‐rational mutagenesis approach, we have developed two RebH variants with expanded biocatalytic repertoires that can efficiently halogenate several novel indole substrates and produce important pharmaceutical intermediates. Interestingly, the engineered enzymes are completely inactive towards their natural substrate tryptophan in spite of their high tolerance to various functional groups in the indole ring. Computational modelling and molecular dynamics simulations provide mechanistic insights into the role of gatekeeper residues in the substrate binding site and the dramatic switch in substrate specificity when these are mutated., Two engineered halogenase enzymes were developed by switching substrate specificity of the parental enzyme from tryptophan to indole compounds. They can halogenate several industrially important indole derivatives more efficiently than the parental enzyme; this includes enzymatic chlorination/bromination of novel substrates that were not reported previously. The engineered enzymes could be useful in regioselective synthesis of drug intermediates.

The discarding and burning of corn stalks in the fields after harvesting lead to environmental pollution and waste of resources. Composting is an effective way to disposal of the crop straws. Composting is a complex biochemical process and needs a detailed study in cold region. Hence, the succession process of bacteria and Actinomycetes in the process of corn stalk composting in cold region was studied by 16SrRNA. Alpha diversity analysis showed that the detection results could represent the real situation. The bacterial community diversity from high to low was F50 > F90 > F0 > F10 > F20. The results of beta analysis showed that F20 and F50 had the most similar microbial structure at the phylum level, and the difference between F0 and F20 was the largest. The dominant microbes changed from Proteobacteria and Bacteroidetes in F0 in heating stage to Firmicutes and Proteobacteria, Actinobacteria and Firmicutes in F10 during early high temperature stage, and Actinobacteria, Proteobacteria and Bacteroidetes in cooling and post composting phases. Actinobacteria and Firmicutes were the dominant bacteria in the whole composting process. In the composting process, the microbial community was mainly involved in amino acid metabolism related to nitrogen transformation and carbohydrate metabolism related to lignocellulose degradation. Lignin and hemicellulose were mainly degraded in thermophilic stage. The conversion of nitrogen and degradation of cellulose occurred mainly in the early stages of composting. The research will be helpful to understand the biochemical process of composting in cold region.

Heptoses are important structural components of Gram-negative bacterium cell wall and participate in bacterial colonization, infection, and immune recognition. Current knowledge of NDP-heptose originating from d-sedoheptulose 7-phosphate in Grampositive bacterium remains limited. Here, in silico analysis suggested that the special tridomain NDP-heptose synthetases with isomerase, kinase, and nucleotidyltransferase activities are conservatively distributed in Actinobacteria class of Gram-positive bacterium. Enzymatical characterization of the tridomain proteins from different strains showed that they are involved in ADP-d-glycero-β-d-manno-heptose biosynthesis despite the unexpected discovery of kinase activities deficient in some proteins. The presence of three types of NDP-heptose synthetases in Gram-positive bacterium suggests that it is also a rich source of heptoses and the heptose moieties may play important roles in vivo. Our work updates the understanding of NDP-heptose biosynthesis in Gram-positive bacterium and lays a solid foundation for further physiological function explorations. Supporting Information The supporting information is available online at 10.1007/s11427-021-2000-2. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

The ecological restoration of coal gangue can be achieved by planting Cajanus cajan (pigeon pea) because of its developed root system. The close relationships soil microorganisms have with plants are crucial for improving soil composition; the soil composition affects nutrient absorption. The microbial composition and function of soil planted with C. cajan in reclaimed land were compared with soil that was not planted with C. cajan (the control). Results showed that the dominant microflora in the soil significantly changed after planting C. cajan. Before planting, the dominant microflora included members of the phyla Sulfobacteria and Acidobacteria. After planting, the dominant microflora contained bacteria from phyla and classes that included Actinobacteria, Acidimicubia, Thermoleophilia, and Anaerolineae. Additionally, there were significant differences in the bacterial composition of each layer in soils planted with C. cajan. Principal component analysis revealed that the interpretation degrees of the results for PC2 and PC3 axes were 10.46% and 3.87%, respectively. The dominant microflora were Vicinamibacterales, Nocardioides, and Arthrobacter in the surface soil; Actinophytocola and Sphingomonas in the deep soil; and Sulfobacillus and Acidimicrobium in the mixed-layer soil. Function prediction analysis using the bioinformatics software package PICRUSt revealed that the abundance of operational taxonomic units corresponding to sigma 54-specific transcriptional regulators, serine threonine protein kinase, and histidine kinase increased by 111.2%, 56.8%, and 47.4%, respectively, after planting C. cajan. This study provides a reference for interactions among microorganisms in reclaimed soils for guiding the development and restoration of waste coal gangue hills.

Short-chained aliphatic polyamines (PAs) have recently been recognized as an important carbon, nitrogen, and/or energy source for marine bacterioplankton. To study the genes and taxa involved in the transformations of different PA compounds and their potential variations among marine systems, we collected surface bacterioplankton from nearshore, offshore, and open ocean stations in the Gulf of Mexico and examined their metagenomic responses to additions of single PA model compounds (putrescine, spermidine, or spermine). Genes affiliated with PA uptake and all three known PA degradation pathways, i.e., transamination, γ-glutamylation, and spermidine cleavage, were significantly enriched in most PA-treated metagenomes. In addition, identified PA-transforming taxa were mostly the alpha and gamma classes of Proteobacteria, with less important contributions from members of Betaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and Planctomycetes. These findings suggest that PA transformations are ubiquitous, have diverse pathways, and are carried out by a broad range of the bacterioplankton taxa in the Gulf of Mexico. Identified PA-transforming bacterial genes and taxa were different among nearshore, offshore, and open ocean sites, but were little different among individual compound-amended metagenomes at any specific site. These observations further indicate that PA-transforming taxa and genes are site-specific and with high similarities among PA compounds. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42995-021-00114-x.

Strain M2T was isolated from the beach of Cuxhaven, Wadden Sea, Germany, in course of a program to attain new producers of bioactive natural products. Strain M2T produces litoralimycin and sulfomycin-type thiopeptides. Bioinformatic analysis revealed a potential biosynthetic gene cluster encoding for the M2T thiopeptides. The strain is Gram-stain-positive, rod shaped, non-motile, spore forming, showing a yellow colony color and forms extensively branched substrate mycelium and aerial hyphae. Inferred from the 16S rRNA gene phylogeny strain M2T affiliates with the genus Streptomonospora. It shows 96.6% 16S rRNA gene sequence similarity to the type species Streptomonospora salina DSM 44593 T and forms a distinct branch with Streptomonospora sediminis DSM 45723 T with 97.0% 16S rRNA gene sequence similarity. Genome-based phylogenetic analysis revealed that M2T is closely related to Streptomonospora alba YIM 90003 T with a digital DNA-DNA hybridisation (dDDH) value of 26.6%. The predominant menaquinones of M2T are MK-10(H6), MK-10(H8), and MK-11(H6) (> 10%). Major cellular fatty acids are iso-C16:0, anteiso C17:0 and C18:0 10-methyl. The polar lipid profile consisted of diphosphatidylglycerol phosphatidyl glycerol, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, three glycolipids, two unknown phospholipids, and two unknown lipids. The genome size of type strain M2T is 5,878,427 bp with 72.1 mol % G + C content. Based on the results obtained from phylogenetic and chemotaxonomic studies, strain M2T (= DSM 106425 T = NCCB 100650 T) is considered to represent a novel species within the genus Streptomonospora for which the name Streptomonospora litoralis sp. nov. is proposed.

Crude oil extracted from oilfield reservoirs brings together hypersaline produced water. Failure in pipelines transporting this mixture causes contamination of the soil with oil and hypersaline water. Soil salinization is harmful to biological populations, impairing the biodegradation of contaminants. We simulated the contamination of a soil from an oilfield with produced water containing different concentrations of NaCl and crude oil, in order to evaluate the effect of salinity and hydrocarbon concentration on prokaryote community structure and biodegradation activity. Microcosms were incubated in CO(2)-measuring respirometer. After the incubation, residual aliphatic hydrocarbons were quantified and were performed 16S rRNA gene sequencing. An increase in CO(2) emission and hydrocarbon biodegradation was observed with increasing oil concentration up to 100 g kg(−1). Alpha diversity decreased in oil-contaminated soils with an increase in the relative abundance of Actinobacteria and reduction of Bacteroidetes with increasing oil concentration. In the NaCl-contaminated soils, alpha diversity, CO(2) emission, and hydrocarbon biodegradation decreased with increasing NaCl concentration. There was an increase in the relative abundance of Firmicutes and Proteobacteria and a reduction of Actinobacteria with increasing salt concentration. Our results highlight the need to adopt specific bioremediation strategies in soils impacted by mixtures of crude oil and hypersaline produced water. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00476-5.

Poultry litter is widely applied as agricultural fertilizer and can affect the soil microbiome through nutrient overload and antibiotic contamination. In this study, we assessed changes in soil bacterial diversity using high-throughput sequencing approaches. Four samples in triplicate were studied: soils with short- and long-term fertilization by poultry litter (S1 = 10 months and S2 = 30 years, respectively), a soil inside a poultry shed (S3), and a forest soil used as control (S0). Samples S0, S1, and S2 revealed a relatively high richness, with confirmed operational taxonomic units (OTUs) in the three replicates of each sample ranging from 1243 to 1279, while richness in S3 was about three times lower (466). The most abundant phyla were Proteobacteria, Bacteroidetes, and Actinobacteria. Acidobacteria, Planctomycetes, and Verrucomicrobia were also abundant but highly diminished in S3, while Firmicutes was less abundant in S0. Changes in bacterial communities were very evident at the genera level. The genera Gaiella, Rhodoplanes, Solirubacter, and Sphingomonas were predominant in S0 but strongly decreased in the other soils. Pedobacter and Devosia were the most abundant in S1 and were diminished in S2, while Herbiconiux, Brevundimonas, Proteiniphilum, and Petrimonas were abundant in S2. The most abundant genera in S3 were Deinococcus, Truepera, Rhodanobacter, and Castellaniella. A predictive analysis of the metabolic functions with Tax4Fun2 software suggested the potential presence of enzymes associated with antibiotic resistance as well as with denitrification pathways, indicating that the S3 soil is a potential source of nitrous oxide, a powerful greenhouse gas. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00437-y.

Coastal sands are important natural recreational facilities that have become hotspots for tourism and economic development. However, these sands harbour diverse microbial assemblages that play a critical role in the balance between public health and ecology. In this study, targeted high-throughput sequencing analysis was used to identify sand-borne bacterial populations at four public beaches in Durban. The effect of heavy metal in shaping the distribution of bacterial metacommunities was determined using canonical correspondence analysis (CCA), while the functional gene profiles were predicted using PICRUSt2 analysis. Sequences matching those of the bacterial phylum Proteobacteria were the most abundant in all samples, followed by those of the phyla Firmicutes, Actinobacteria, Bacteroidetes, and Gemmatimonadetes. Genus-level taxonomic analysis showed the presence of 1163 bacterial genera in all samples combined. The distribution of bacterial communities was shaped by heavy metal concentrations, with the distribution of Flavobacteria, Bacteroidia, and Deltaproteobacteria influenced by Pb and Zn, while B and Cr influenced the distribution of Clostridia and Gammaproteobacteria, respectively. Identified antibiotic resistance genes included the peptidoglycan biosynthesis gene II, III, IV, and V, as well as the polymyxin resistance gene, while the virulence genes included the sitA, fimB, aerobactin synthase, and pilL gene. Our findings demonstrate that beach sand-borne bacteria are reservoirs of virulence and antibiotic resistance genes. Contamination of beach sands with heavy metals selects for both heavy metal resistance and antibiotic resistance in beach sand bacterial communities. Children and immunocompromised people engaging in recreational activities on beaches may be exposed to higher risk of infection.

Frankia sp. strains CgS1, CcI156 and CgMI4 were isolated from Casuarina glauca and C. cunninghamiana nodules. Here, we report the 5.26-, 5.33- and 5.20-Mbp draft genome sequences of Frankia sp. strains CgS1, CcI156 and CgMI4, respectively. Analysis of the genome revealed the presence of high numbers of secondary metabolic biosynthetic gene clusters.

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150–200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units—which reduce chain mobility—PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11–14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy. Computer-aided engineering produces improvements to an enzyme that breaks down poly(ethylene terephthalate) (PET) into its constituent monomers, which are used to synthesize PET of near-petrochemical grade that can be further processed into bottles.

Foam potential and viscometer ramp tests (VRTs) were conducted for three municipal wastewater treatment plants to determine if these methods can relate to mechanisms of foaming to physical and biological constituents in sludge. At all plants, digester volatile solids (VS) concentration correlated (R2 > 0.41) with increases in plastic viscosity, a VRT parameter corresponding to foaming risk. Plastic viscosity also correlated with foam-causing bacteria Gordonia (R2 = 0.38). Foam potential test values increased with Microthrix parvicella (R2> 0.28). For one plant, suspected foam-causing bacteria Mycobacterium negatively correlated with parameters representing foam risk. Microscopic filament counting correlated (R2 = 0.97) with quantitative polymerase chain reaction (qPCR) for Gordonia, suggesting that the more accessible counting method can reliably quantify foam-causing bacteria. Foam potential tests and VRTs resulted in plant-specific correlations with foam-related constituents. Therefore, these tests may provide useful evidence when investigating causes of digester foam events.

The genome of Streptomyces scabies, the predominant causal agent of potato common scab, encodes a potential cutinase, the protein Sub1, which was previously shown to be specifically induced in the presence of suberin. The sub1 gene was expressed in Escherichia coli and the recombinant protein Sub1 was purified and characterized. The enzyme was shown to be versatile because it hydrolyzes a number of natural and synthetic substrates. Sub1 hydrolyzed p-nitrophenyl esters, with the hydrolysis of those harboring short carbon chains being the most effective. The Vmax and Km values of Sub1 for p-nitrophenyl butyrate were 2.36 mol g–1 min–1 and 5.7 10–4 M, respectively. Sub1 hydrolyzed the recalcitrant polymers cutin and suberin because the release of fatty acids from these substrates was observed following the incubation of the enzyme with these polymers. Furthermore, the hydrolyzing activity of the esterase Sub1 on the synthetic polymer polyethylene terephthalate (PET) was demonstrated by the release of terephthalic acid (TA). Sub1 activity on PET was markedly enhanced by the addition of Triton and was shown to be stable at 37°C for at least 20 d.

In the present study, we studied the distribution of silicate mineral weathering bacteria (SWB) in stressed environments that release potassium from insoluble source of mineral. Out of 972 isolates, 340 isolates were positive and mineral weathering potential ranged from 5.55 to 180.05%. Maximum abundance of SWB occurred 44.71% in saline environment followed by 23.53% in low temperature and 12.35% each in high temperature and moisture deficit. Among isolates, silicate mineral weathering efficiency ranged from 1.9 to 72.8 μg mL(−1) available K in liquid medium. The phylogenetic tree of SWB discriminated in three clusters viz. Firmicutes, Proteobacteria and Actinobacteria. This is the first report on SWB in stressed environments and identified 27 genera and 67 species which is not reported earlier. Among them Bacillus was the predominant genera (58.60%) distantly followed by Pseudomonas (6.37%), Staphylococcus (5.10%) and Paenibacillus (4.46%). These bacterial strains could be developed as inoculants for biological replenishment of K in stressed soils. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00210-2) contains supplementary material, which is available to authorized users.

The active vitamin D metabolites 25‐OH−D and 1α,25‐(OH)2−D play an essential role in controlling several cellular processes in the human body and are potentially effective in the treatment of several diseases, such as autoimmune diseases, cardiovascular diseases and cancer. The microbial synthesis of vitamin D2 (VD2) and vitamin D3 (VD3) metabolites has emerged as a suitable alternative to established complex chemical syntheses. In this study, a novel strain, Kutzneria albida, with the ability to form 25‐OH−D2 and 25‐OH−D3 was identified. To further improve the conversion of the poorly soluble substrates, several solubilizers were tested. 100‐fold higher product concentrations of 25‐OH−D3 and tenfold higher concentrations of 25‐OH−D2 after addition of 5 % (w/v) 2‐hydroxypropyl β‐cyclodextrin (2‐HPβCD) were reached. Besides the single‐hydroxylation products, the human double‐hydroxylation products 1,25‐(OH)2−D2 and 1,25‐(OH)2−D3 and various other potential single‐ and double‐hydroxylation products were detected. Thus, K. albida represents a promising strain for the biotechnological production of VD2 and VD3 metabolites., The microbial transformation of vitamin D to the active forms by a two‐step hydroxylation represents a promising alternative to complex chemical syntheses. The ascomycete K. albida was shown to catalyze vitamin D hydroxylation by forming diverse single‐ and double‐hydroxylation products. The efficiency of the reaction could be drastically increased by the addition of cyclodextrin derivatives to the biotransformation.

Leifsonia xyli subsp. xyli (Lxx) colonizes the xylem vessels of sugarcane, a plant niche where microorganisms are highly exposed to oxidative and osmotic stresses. This study performed an in silico analysis of the genome of Lxx and characterized 16 genes related to the detoxification of oxidative species (peroxidases, O(2)(−) dismutases, and methionine reductases) and to the production and transport of osmolytes and analyzed their expression in vitro after 30, 60, and 120 min of exposure to H(2)O(2) or PEG. The PAGE activity of superoxide dismutase (Mn-SOD as confirmed by inhibition tests) and of catalase (CAT) and the accumulation of trehalose were also assessed. Exposure to H(2)O(2) increased the expression of most oxidative-responsive genes and decreased the expression of those related to osmotic responses, whereas the opposite occurred after exposure to PEG. The isoform profiles of CAT and Mn-SOD shifted in response to H(2)O(2) but not to PEG and Lxx cells accumulated more trehalose over time after exposure to PEG compared with non-exposed cells. The experimental results validated the in silico analysis and indicated that this obligate endophytic parasite has multiple and functional mechanisms to combat the stresses imposed by its host. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00163-6) contains supplementary material, which is available to authorized users.

Distillery sludge is a major source of aquatic pollution, but little is known about their microbial community and their association with the organic and metal pollutants. Sugarcane molasses-based distillery is an important industry in India, although the waste is usually treated prior to disposal, the treatment is often inadequate. The adverse effects of the organic and metal pollutants in sugarcane molasses-based distillery sludge on the microbial biodiversity and abundance in the disposal site have not been elucidated. This study aims to address this gap of knowledge. Samples were collected from the discharge point, 1 and 2 km downstream (D1, D2, and D3, respectively) of a sugarcane distillery in Uttar Pradesh, India, and their physico-chemical properties characterised. Using QIIME, taxonomic assignment for the V3 and V4 hypervariable regions of 16 S rRNA was performed. The phyla Proteobacteria (28–39%), Firmicutes (20–28%), Bacteriodetes (9–10%), Actinobacteria (5–10%), Tenericutes (1–9%) and Patescibacteria (2%) were the predominant bacteria in all three sites. Euryechaeota, were detected in sites D1 and D2 (1–2%) but absent in D3. Spirochaetes (5%), Sinergistetes (2%) and Cloacimonetes (1%) were only detected in samples from site D1. Shannon, Simpson, Chao1, and Observed-species indices indicated that site D1 (10.18, 0.0013, 36706.55 and 45653.84, respectively) has higher bacterial diversity and richness than D2 (6.66, 0.0001, 25987.71 and 49655.89, respectively) and D3 (8.31, 0.002, 30345.53 and 30654.88, respectively), suggesting the organic and metal pollutants provided the stressors to favour the survival of microbial community that can biodegrade and detoxify them in the distillery sludge. This study confirmed that the treatment of the distillery waste was not sufficiently effective and provided new metagenomic information on its impact on the surrounding microbial community. It also offered new insights into potential bioremediation candidates.

Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R‐selective ATAs belong to the fold type IV PLP‐dependent enzymes, and different sequence‐, structure‐ and substrate scope‐based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)‐ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)‐ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)‐ATAs. The ATA from Exophiala sideris contains a motif characteristic for d‐ATAs, which was previously believed to be a disqualifying factor for (R)‐ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram‐negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)‐ATAs with a shortened/missing N‐terminal helix. The active‐site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope., Diversifying: The toolbox of R‐selective amine transaminases (ATAs) has been expanded by an ATA containing a motif characteristic for d‐ATAs, two ATAs with a shortened/missing N‐terminal helix and the first ATA with known structure from a Gram‐negative bacterium. Their active site charges vary significantly compared to known ATAs correlating with their diverging substrate scope.

Five new members of the salinilactone family, salinilactones D–H, are reported. These bicyclic lactones are produced by Salinispora bacteria and display extended or shortened alkyl side chains relative to the recently reported salinilactones A–C. They were identified by GC/MS, gas chromatographic retention index, and comparison with synthetic samples. We further investigated the occurrence of salinilactones across six newly proposed Salinispora species to gain insight into how compound production varies among taxa. The growth‐inhibiting effect of this compound family on multiple biological systems including non‐Salinispora actinomycetes was analyzed. Additionally, we found strong evidence for significant cytotoxicity of the title compounds., Finding further function: Five new salinilactones have been identified by GC/MS and synthesis, thereby extending the family of salinilactones to eight compounds. Inhibition assays with actinomycetes, tracking of salinilactone production in Salinispora bacteria, and brine shrimp assays provided insight into the occurrence and biological purpose of salinilactones.

Rubber is an essential part of our daily lives with thousands of rubber-based products being made and used. Natural rubber undergoes chemical processes and structural modifications, while synthetic rubber, mainly synthetized from petroleum by-products are difficult to degrade safely and sustainably. The most prominent group of biological rubber degraders are Actinobacteria. Rubber degrading Actinobacteria contain rubber degrading genes or rubber oxygenase known as latex clearing protein (lcp). Rubber is a polymer consisting of isoprene, each containing one double bond. The degradation of rubber first takes place when lcp enzyme cleaves the isoprene double bond, breaking them down into the sole carbon and energy source to be utilized by the bacteria. Actinobacteria grow in diverse environments, and lcp gene containing strains have been detected from various sources including soil, water, human, animal, and plant samples. This review entails the occurrence, physiology, biochemistry, and molecular characteristics of Actinobacteria with respect to its rubber degrading ability, and discusses possible technological applications based on the activity of Actinobacteria for treating rubber waste in a more environmentally responsible manner.

In 2017, the 560-ha area of hybrid poplar plantation in northern Poland showed symptoms of tree decline. Leaves appeared smaller, turned yellow-brown, and were shed prematurely. Twigs and smaller branches died. Bark was sunken and discolored, often loosened and split. Trunks decayed from the base. Phloem and xylem showed brown necrosis. Ten per cent of trees died in 1–2 months. None of these symptoms was typical for known poplar diseases. Bacteria in soil and the necrotic base of poplar trunk were analysed with Illumina sequencing. Soil and wood were colonized by at least 615 and 249 taxa. The majority of bacteria were common to soil and wood. The most common taxa in soil were: Acidobacteria (14.757%), Actinobacteria (14.583%), Proteobacteria (36.872) with Betaproteobacteria (6.516%), Burkholderiales (6.102%), Comamonadaceae (2.786%), and Verrucomicrobia (5.307%).The most common taxa in wood were: Bacteroidetes (22.722%) including Chryseobacterium (5.074%), Flavobacteriales (10.873%), Sphingobacteriales (9.396%) with Pedobacter cryoconitis (7.306%), Proteobacteria (73.785%) with Enterobacteriales (33.247%) including Serratia (15.303%) and Sodalis (6.524%), Pseudomonadales (9.829%) including Pseudomonas (9.017%), Rhizobiales (6.826%), Sphingomonadales (5.646%), and Xanthomonadales (11.194%). Possible pathogens were Pseudomonas, Rhizobium and Xanthomonas. The initially endophytic character of bacteria is emphasized. Soil and possibly planting material might be the sources of pathogen inoculum.

Central-Andean Ecosystems (between 2000 and 6000 m above sea level (masl) are typical arid-to-semiarid environments suffering from the highest total solar and ultraviolet-B radiation on the planet but displaying numerous salt flats and shallow lakes. Andean microbial ecosystems isolated from these environments are of exceptional biodiversity enduring multiple severe conditions. Furthermore, the polyextremophilic nature of the microbes in such ecosystems indicates the potential for biotechnological applications. Within this context, the study undertaken used genome mining, physiological and microscopical characterization to reveal the multiresistant profile of Nesterenkonia sp. Act20, an actinobacterium isolated from the soil surrounding Lake Socompa, Salta, Argentina (3570 masl). Ultravioet-B, desiccation, and copper assays revealed the strain’s exceptional resistance to all these conditions. Act20’s genome presented coding sequences involving resistance to antibiotics, low temperatures, ultraviolet radiation, arsenic, nutrient-limiting conditions, osmotic stress, low atmospheric-oxygen pressure, heavy-metal stress, and toxic fluoride and chlorite. Act20 can also synthesize proteins and natural products such as an insecticide, bacterial cellulose, ectoine, bacterial hemoglobin, and even antibiotics like colicin V and aurachin C. We also found numerous enzymes for animal- and vegetal-biomass degradation and applications in other industrial processes. The resilience of Act20 and its biotechnologic potential were thoroughly demonstrated in this work. Supplementary Information The online version contains supplementary material available at 10.1007/s11274-021-03129-9

Structural and enzymological explorations of p-hydroxy-mandelate oxidase and its mutants uncover an unprecedented electrophilic/nucleophilic duality for the flavin mononucleotide cofactor as well as an intramolecular disproportionation mechanism for an oxidative decarboxylation reaction., The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportion­ation reaction via an N5-alkanol-FMNred C′α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.

A set of oligonucleotide primers, Rubro223f and Rubro454r, were found to amplify a 267 nucleotide sequence of 16S rRNA genes of Rubrobacter type strains. The primers distinguished members of this genus from other deeply-rooted actinobacterial lineages corresponding to the genera Conexibacter, Gaiella, Parviterribacter, Patulibacter, Solirubrobacter and Thermoleophilum of the class Thermoleophilia. Amplification of DNA bands of about 267 nucleotides were generated from environmental DNA extracted from soil samples taken from two locations in the Atacama Desert. Sequencing of a DNA library prepared from the bands showed that all of the clones fell within the evolutionary radiation occupied by the genus Rubrobacter. Most of the clones were assigned to two lineages that were well separated from phyletic lines composed of Rubrobacter type strains. It can be concluded that primers Rubro223f and Rubro454r are specific for the genus Rubrobacter and can be used to detect the presence and abundance of members of this genus in the Atacama Desert and other biomes. Electronic supplementary material The online version of this article (10.1007/s10482-019-01314-3) contains supplementary material, which is available to authorized users.

The occurrence of pests and diseases can affect plant health and productivity in ecosystems that are already at risk, such as tropical montane cloud forests. The use of naturally occurring microorganisms is a promising alternative to mitigate forest tree fungal pathogens. The objectives of this study were to isolate rhizobacteria associated with five Lauraceae species from a Mexican tropical montane cloud forest and to evaluate their antifungal activity against Fusarium solani and F. oxysporum. Fifty-six rhizobacterial isolates were assessed for mycelial growth inhibition of Fusarium spp. through dual culture assays. Thirty-three isolates significantly reduced the growth of F. solani, while 21 isolates inhibited that of F. oxysporum. The nine bacterial isolates that inhibited fungal growth by more than 20% were identified through 16S rDNA gene sequence analysis; they belonged to the genera Streptomyces, Arthrobacter, Pseudomonas, and Staphylococcus. The volatile organic compounds (VOC) produced by these nine isolates were evaluated for antifungal activity. Six isolates (Streptomyces sp., Arthrobacter sp., Pseudomonas sp., and Staphylococcus spp.) successfully inhibited F. solani mycelial growth by up to 37% through VOC emission, while only the isolate INECOL-21 (Pseudomonas sp.) inhibited F. oxysporum. This work provides information on the microbiota of Mexican Lauraceae and is one of the few studies identifying forest tree–associated microbes with inhibitory activity against tree pathogens. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00094-2) contains supplementary material, which is available to authorized users.

In recent years, there has been an increasing interest in the remediation of contaminated environments, and a suitable solution is in situ bioremediation. To achieve this, large-scale bacterial biomass production should be sustainable, using economic culture media. The main aim of this study was to optimize the physicochemical conditions for the biomass production of an actinobacterium with well-known bioremediation ability using inexpensive substrates and to scale-up its production in a bioreactor. For this, the growth of four strains of actinobacteria were evaluated in minimal medium with glucose and glycerol as carbon and energy sources. In addition, l-asparagine and ammonium sulfate were assayed as alternative nitrogen sources. The strain Streptomyces sp. A5 showed the highest biomass production in shake-flasks culture using glycerol and ammonium sulfate as carbon and nitrogen sources, respectively. Factorial designs with five factors (glycerol concentration, inoculum size, pH, temperature, and agitation) were employed to optimize the biomass production of Streptomyces sp. A5. The maximum biomass production was obtained using 5 g L(−1) of glycerol, 0.25 µL of inoculum, pH 7, 30 °C and 200 rpm. Finally, the production was successfully scaled to a 2 L stirred tank bioreactor. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02588-5.

Bile acids are detergent molecules that solubilize dietary lipids and lipid-soluble vitamins. Humans synthesize bile acids with α-orientation hydroxyl groups which can be biotransformed by gut microbiota to toxic, hydrophobic bile acids, such as deoxycholic acid (DCA). Gut microbiota can also convert hydroxyl groups from the α-orientation through an oxo-intermediate to the β-orientation, resulting in more hydrophilic, less toxic bile acids. This interconversion is catalyzed by regio- (C-3 vs. C-7) and stereospecific (α vs. β) hydroxysteroid dehydrogenases (HSDHs). So far, genes encoding the urso- (7α-HSDH & 7β-HSDH) and iso- (3α-HSDH & 3β-HSDH) bile acid pathways have been described. Recently, multiple human gut clostridia were reported to encode 12α-HSDH, which interconverts DCA and 12-oxolithocholic acid (12-oxoLCA). 12β-HSDH completes the epi-bile acid pathway by converting 12-oxoLCA to the 12β-bile acid denoted epiDCA; however, a gene(s) encoding this enzyme has yet to be identified. We confirmed 12β-HSDH activity in cultures of Clostridium paraputrificum ATCC 25780. From six candidate C. paraputrificum ATCC 25780 oxidoreductase genes, we discovered the first gene (DR024_RS09610) encoding bile acid 12β-HSDH. Phylogenetic analysis revealed unforeseen diversity for 12β-HSDH, leading to validation of two additional bile acid 12β-HSDHs through a synthetic biology approach. By comparison to a previous phylogenetic analysis of 12α-HSDH, we identified the first potential C-12 epimerizing strains: Collinsella tanakaei YIT 12063 and Collinsella stercoris DSM 13279. A Hidden Markov Model search against human gut metagenomes located putative 12β-HSDH genes in about 30% of subjects within the cohorts analyzed, indicating this gene is relevant in the human gut microbiome.

Microbial rhodopsins, comprising a protein moiety (rhodopsin apoprotein) bound to the light-absorbing chromophore retinal, function as ion pumps, ion channels, or light sensors. However, recent genomic and metagenomic surveys showed that some rhodopsin-possessing prokaryotes lack the known genes for retinal biosynthesis. Since rhodopsin apoproteins cannot absorb light energy, rhodopsins produced by prokaryotic strains lacking genes for retinal biosynthesis are hypothesized to be non-functional in cells. In the present study, we investigated whether Aurantimicrobium minutum KNCT, which is widely distributed in terrestrial environments and lacks any previously identified retinal biosynthesis genes, possesses functional rhodopsin. We initially measured ion transport activity in cultured cells. A light-induced pH change in a cell suspension of rhodopsin-possessing bacteria was detected in the absence of exogenous retinal. Furthermore, spectroscopic analyses of the cell lysate and HPLC-MS/MS analyses revealed that this strain contained an endogenous retinal. These results confirmed that A. minutum KNCT possesses functional rhodopsin and, hence, produces retinal via an unknown biosynthetic pathway. These results suggest that rhodopsin-possessing prokaryotes lacking known retinal biosynthesis genes also have functional rhodopsins.

Seven emblematic Leonardo da Vinci’s drawings were investigated through third generation sequencing technology (Nanopore). In addition, SEM analyses were carried out to acquire photographic documentation and to infer the nature of the micro-objects removed from the surface of the drawings. The Nanopore generated microbiomes can be used as a “bio-archive” of the drawings, offering a kind of fingerprint for current and future biological comparisons. This information might help to create a biological catalog of the drawings (cataloging), a microbiome-fingerprint for each single analyzed drawing, as a reference dataset for future studies (monitoring) and last but not least a bio-archive of the history of each single object (added value). Results showed a relatively high contamination with human DNA and a surprising dominance of bacteria over fungi. However, it was possible to identify typical bacteria of the human microbiome, which are mere contaminants introduced by handling of the drawings as well as other microorganisms that seem to have been introduced through vectors, such as insects and their droppings, visible through the SEM analyses. All drawings showed very specific bio-archives, but a core microbiome of bacteria and fungi that are repeatedly found in this type of material as true degraders were identified, such as members of the phyla Proteobacteria, Actinobacteria, and Firmicutes among bacteria, and fungi belonging to the classes Sordariomycetes and Eurotiomycetes. In addition, some similarities were observed that could be influenced by their geographical location (Rome or Turin), indicating the influence of this factor and denoting the importance of environmental and storage conditions on the specific microbiomes.

Emerging and re-emerging microbial pathogens, together with their rapid evolution and adaptation against antibiotics, highlight the importance not only of screening for new antimicrobial agents, but also for deepening knowledge about existing antibiotics. Primycin is a large 36-membered non-polyene macrolide lactone exclusively produced by Saccharomonospora azurea. This study provides information about strain dependent primycin production ability in conjunction with the structural, functional and comparative genomic examinations. Comparison of high- and low-primycin producer strains, transcriptomic analysis identified a total of 686 differentially expressed genes (DEGs), classified into diverse Cluster of Orthologous Groups. Among them, genes related to fatty acid synthesis, self-resistance, regulation of secondary metabolism and agmatinase encoding gene responsible for catalyze conversion between guanidino/amino forms of primycin were discussed. Based on in silico data mining methods, we were able to identify DEGs whose altered expression provide a good starting point for the optimization of fermentation processes, in order to perform targeted strain improvement and rational drug design. Electronic supplementary material The online version of this article (10.1007/s11274-020-02935-x) contains supplementary material, which is available to authorized users.

The petrochemical industry is responsible for many accidental releases of pollutants in soil such as hydrocarbons and toxic metals. This co-contamination is responsible for a delay in the degradation of the organic pollution. Many successful technologies to remove these metals apply extracellular polymeric substances (EPS). In this study, we tested the application of an EPS from a Paenibacillus sp. to aid the bioremediation of soils contaminated with crude oil and nickel. We conducted a microcosm experiment to soils containing combinations of oil, nickel, and EPS. The final concentration of oil was evaluated with an infrared spectrometer. Also, we sequenced the metagenomes of the samples in an ion torrent sequencer. The application of EPS did not aid the removal of hydrocarbons with or without the presence of nickel. However, it led to a smaller decrease in the diversity indexes. EPS decreased the abundance of Actinobacteria and increased that of Proteobacteria. The EPS also decreased the connectivity among Actinobacteria in the network analysis. The results indicated that the addition of EPS had a higher effect on the community structure than nickel. Altogether, our results indicate that this approach did not aid the bioremediation of hydrocarbons likely due to its effect in the community structure that affected hydrocarbonoclastic microorganisms. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00428-z.

INTRODUCTION: Freshwater ecosystems provide propitious conditions for the acquisition and spread of antibiotic resistance genes (ARGs), and integrons play an important role in this process. MATERIAL AND METHODS: In the present study, the diversity of putative environmental integron-cassettes, as well as their potential bacterial hosts in the Velhas River (Brazil), was explored through intI-attC and 16S rRNA amplicons deep sequencing. RESULTS AND DISCUSSION: ORFs related to different biological processes were observed, from DNA integration to oxidation-reduction. ARGs-cassettes were mainly associated with class 1 mobile integrons carried by pathogenic Gammaproteobacteria, and possibly sedentary chromosomal integrons hosted by Proteobacteria and Actinobacteria. Two putative novel ARG-cassettes homologs to fosB3 and novA were detected. Regarding 16SrRNA gene analysis, taxonomic and functional profiles unveiled Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria as dominant phyla. Betaproteobacteria, Alphaproteobacteria, and Actinobacteria classes were the main contributors for KEGG orthologs associated with resistance. CONCLUSIONS: Overall, these results provide new information about environmental integrons as a source of resistance determinants outside clinical settings and the bacterial community in the Velhas River. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-020-00409-8.