Isolation and characterization of Dehalobacter sp. strain TeCB1 including identification of TcbA: A novel tetra- and trichlorobenzene reductive Dehalogenase

© 2017 Alfán-Guzmán, Ertan, Manefield and Lee. Dehalobacter sp. strain TeCB1 was isolated from groundwater near Sydney, Australia, that is polluted with a range of organochlorines. The isolated strain is able to grow by reductive dechlorination of 1,2,4,5-tetrachlorobenzene to 1,3- and 1,4-dichlorobenzene with 1,2,4-trichlorobenzene being the intermediate daughter product. Transient production of 1,2-dichlorobenzene was detected with subsequent conversion to monochlorobenzene. The dehalogenation capability of strain TeCB1 to respire 23 alternative organochlorines was examined and shown to be limited to the use of 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene. Growth on 1,2,4-trichlorobenzene resulted in the production of predominantly 1,3- and 1,4-dichlorobenzene. The inability of strain TeCB1 to grow on 1,2-dichlorobenzene indicated that the production of monochlorobenzene during growth on 1,2,4,5-tetarchlorobezene was cometabolic. The annotated genome of strain TeCB1 contained only one detectable 16S rRNA gene copy and genes for 23 full-length and one truncated Reductive Dehalogenase (RDase) homologs, five unique to strain TeCB1. Identification and functional characterization of the 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene RDase (TcbA) was achieved using native-PAGE coupled with liquid chromatography tandem mass spectrometry. Interestingly, TcbA showed higher amino acid identity with tetrachloroethene reductases PceA (95% identity) from Dehalobacter restrictus PER-K23 and Desulfitobacterium hafniense Y51 than with the only other chlorinated benzene reductase [i.e., CbrA (30% identity)] functionally characterized to date

Biodiversity and ecosystem function in soil

1. Soils are one of the last great frontiers for biodiversity research and are home to an extraordinary range of microbial and animal groups. Biological activities in soils drive many of the key ecosystem processes that govern the global system, especially in the cycling of elements such as carbon, nitrogen and phosphorus. 2. We cannot currently make firm statements about the scale of biodiversity in soils, or about the roles played by soil organisms in the transformations of organic materials that underlie those cycles. The recent UK Soil Biodiversity Programme (SBP) has brought a unique concentration of researchers to bear on a single soil in Scotland, and has generated a large amount of data concerning biodiversity, carbon flux and resilience in the soil ecosystem. 3. One of the key discoveries of the SBP was the extreme diversity of small organisms: researchers in the programme identified over 100 species of bacteria, 350 protozoa, 140 nematodes and 24 distinct types of arbuscular mycorrhizal fungi. Statistical analysis of these results suggests a much greater 'hidden diversity'. In contrast, there was no unusual richness in other organisms, such as higher fungi, mites, collembola and annelids. 4. Stable-isotope (C-13) technology was used to measure carbon fluxes and map the path of carbon through the food web. A novel finding was the rapidity with which carbon moves through the soil biota, revealing an extraordinarily dynamic soil ecosystem. 5. The combination of taxonomic diversity and rapid carbon flux makes the soil ecosystem highly resistant to perturbation through either changing soil structure or removing selected groups of organisms

A bacterial chloroform reductive dehalogenase: purification and biochemical characterization

© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. We report herein the purification of a chloroform (CF)-reducing enzyme, TmrA, from the membrane fraction of a strict anaerobe Dehalobacter sp. strain UNSWDHB to apparent homogeneity with an approximate 23-fold increase in relative purity compared to crude lysate. The membrane fraction obtained by ultracentrifugation was solubilized in Triton X-100 in the presence of glycerol, followed by purification by anion exchange chromatography. The molecular mass of the purified TmrA was determined to be 44.5 kDa by SDS-PAGE and MALDI-TOF/TOF. The purified dehalogenase reductively dechlorinated CF to dichloromethane in vitro with reduced methyl viologen as the electron donor at a specific activity of (1.27 ± 0.04) × 103units mg protein−1. The optimum temperature and pH for the activity were 45°C and 7.2, respectively. The UV-visible spectrometric analysis indicated the presence of a corrinoid and two [4Fe-4S] clusters, predicted from the amino acid sequence. This is the first report of the production, purification and biochemical characterization of a CF reductive dehalogenase

Evidence for a Putative Isoprene Reductase in Acetobacterium wieringae

Recent discoveries of isoprene-metabolizing microorganisms suggest they might play an important role in the global isoprene budget. Under anoxic conditions, isoprene can be used as an electron acceptor and is reduced to methylbutene. This study describes the proteogenomic profiling of an isoprene-reducing bacterial culture to identify organisms and genes responsible for the isoprene hydrogenation reaction. A metagenome-assembled genome (MAG) of the most abundant (89% relative abundance) lineage in the enrichment, Acetobacterium wieringae, was obtained. Comparative proteogenomics and reverse transcription-PCR (RT-PCR) identified a putative five-gene operon from the A. wieringae MAG upregulated during isoprene reduction. The operon encodes a putative oxidoreductase, three pleiotropic nickel chaperones (2 × HypA, HypB), and one 4Fe-4S ferredoxin. The oxidoreductase is proposed as the putative isoprene reductase with a binding site for NADH, flavin adenine dinucleotide (FAD), two pairs of canonical [4Fe-4S] clusters, and a putative iron-sulfur cluster site in a Cys6bonding environment. Well-studied Acetobacterium strains, such as A. woodii DSM 1030, A. wieringae DSM 1911, or A. malicum DSM 4132, do not encode the isoprene-regulated operon but encode, like many other bacteria, a homolog of the putative isoprene reductase (;47 to 49% amino acid sequence identity). Uncharacterized homologs of the putative isoprene reductase are observed across the Firmicutes, Spirochaetes, Tenericutes, Actinobacteria, Chloroflexi, Bacteroidetes, and Proteobacteria, suggesting the ability of biohydrogenation of unfunctionalized conjugated doubled bonds in other unsaturated hydrocarbons

Investigation of the microbial communities colonizing prepainted steel used for roofing and walling

© 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. Microbial colonization of prepainted steel, commonly used in roofing applications, impacts their aesthetics, durability, and functionality. Understanding the relevant organisms and the mechanisms by which colonization occurs would provide valuable information that can be subsequently used to design fouling prevention strategies. Here, next-generation sequencing and microbial community finger printing (T-RFLP) were used to study the community composition of microbes colonizing prepainted steel roofing materials at Burrawang, Australia and Kapar, Malaysia over a 52-week period. Community diversity was low and was dominated by Bacillus spp., cyanobacteria, actinobacteria, Cladosporium sp., Epicoccum nigrum, and Teratosphaeriaceae sp. Cultivation-based methods isolated approximately 20 different fungi and bacteria, some of which, such as E. nigrum and Cladosporium sp., were represented in the community sequence data. Fluorescence in situ hybridization imaging showed that fungi were the most dominant organisms present. Analysis of the sequence and T-RFLP data indicated that the microbial communities differed significantly between locations and changed significantly over time. The study demonstrates the utility of molecular ecology tools to identify and characterize microbial communities associated with the fouling of painted steel surfaces and ultimately can enable the targeted development of control strategies based on the dominant species responsible for fouling

Microbiologically influenced corrosion of cable bolts in underground coal mines: The effect of Acidithiobacillus ferrooxidans

Reports on corrosion failure of cable bolts, used in mining and civil industries, have been increasing in the past two decades. The previous studies found that pitting corrosion on the surface of a cable bolt can initiate premature failure of the bolt. In this study, the role of Acidithiobacillus ferrooxidans (A. ferrooxidans) bacterium in the occurrence of pitting corrosion in cable bolts was studied. Stressed coupons, made from the wires of cable bolts, were immersed in testing bottles containing groundwater collected from an underground coal mine and a mixture of A. ferrooxidans and geomaterials. It was observed that A. ferrooxidans caused pitting corrosion on the surface of cable bolts in the near-neutral environment. The presence of geomaterials slightly affected the pH of the environment; however, it did not have any significant influence on the corrosion activity of A. ferrooxidans. This study suggests that the common bacterium A. ferrooxidans found in many underground environments can be a threat to cable bolts’ integrity by creating initiation points for other catastrophic failures such as stress corrosion cracking

Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl backbone as antibacterial agents

© 2017 Elsevier Masson SAS Antimicrobial peptides (AMPs) and their synthetic mimics have received recent interest as new alternatives to traditional antibiotics in attempts to overcome the rise of antibiotic resistance in many microbes. AMPs are part of the natural defenses of most living organisms and they also have a unique mechanism of action against bacteria. Herein, a new series of short amphiphilic cationic peptidomimetics were synthesized by incorporating the 3′-amino-[1,1′-biphenyl]-3-carboxylic acid backbone to mimic the essential properties of natural AMPs. By altering hydrophobicity and charge, we identified the most potent analogue 25g that was active against both Gram-positive Staphylococcus aureus (MIC = 15.6 μM) and Gram-negative Escherichia coli (MIC = 7.8 μM) bacteria. Cytoplasmic permeability assay results revealed that 25g acts primarily by depolarization of lipids in cytoplasmic membranes. The active compounds were also investigated for their cytotoxicity to human cells, lysis of lipid bilayers using tethered bilayer lipid membranes (tBLMs) and their activity against established biofilms of S. aureus and E. coli

Darkness visible: reflections on underground ecology

1 Soil science and ecology have developed independently, making it difficult for ecologists to contribute to urgent current debates on the destruction of the global soil resource and its key role in the global carbon cycle. Soils are believed to be exceptionally biodiverse parts of ecosystems, a view confirmed by recent data from the UK Soil Biodiversity Programme at Sourhope, Scotland, where high diversity was a characteristic of small organisms, but not of larger ones. Explaining this difference requires knowledge that we currently lack about the basic biology and biogeography of micro-organisms. 2 It seems inherently plausible that the high levels of biological diversity in soil play some part in determining the ability of soils to undertake ecosystem-level processes, such as carbon and mineral cycling. However, we lack conceptual models to address this issue, and debate about the role of biodiversity in ecosystem processes has centred around the concept of functional redundancy, and has consequently been largely semantic. More precise construction of our experimental questions is needed to advance understanding. 3 These issues are well illustrated by the fungi that form arbuscular mycorrhizas, the Glomeromycota. This ancient symbiosis of plants and fungi is responsible for phosphate uptake in most land plants, and the phylum is generally held to be species-poor and non-specific, with most members readily colonizing any plant species. Molecular techniques have shown both those assumptions to be unsafe, raising questions about what factors have promoted diversification in these fungi. One source of this genetic diversity may be functional diversity. 4 Specificity of the mycorrhizal interaction between plants and fungi would have important ecosystem consequences. One example would be in the control of invasiveness in introduced plant species: surprisingly, naturalized plant species in Britain are disproportionately from mycorrhizal families, suggesting that these fungi may play a role in assisting invasion. 5 What emerges from an attempt to relate biodiversity and ecosystem processes in soil is our extraordinary ignorance about the organisms involved. There are fundamental questions that are now answerable with new techniques and sufficient will, such as how biodiverse are natural soils? Do microbes have biogeography? Are there rare or even endangered microbes

Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target

YesN-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach.University of Malaya High Impact Research (HIR) Grant (UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/14/1, no. H-50001-A000027) given to K.G.C. and National Natural Science Foundation of China (no. 81260481) given to H.W