Neil L. Wilson

Untangling the multiple monooxygenases of Mycobacterium chubuense strain NBB4, a versatile hydrocarbon degrader

Mycobacterium strain NBB4 was isolated on ethene as part of a bioprospecting study searching for novel monooxygenase (MO) enzymes of interest to biocatalysis and bioremediation. Previous work indicated that strain NBB4 contained an unprecedented diversity of MO genes, and we hypothesized that each MO type would support growth on a distinct hydrocarbon substrate. Here, we attempted to untangle the relationships between MO types and hydrocarbon substrates. Strain NBB4 was shown to grow on C2 -C4 alkenes and C2 -C16 alkanes. Complete gene clusters encoding six different monooxygenases were recovered from a fosmid library, including homologues of ethene MO (etnABCD), propene MO (pmoABCD), propane MO (smoABCD), butane MO (smoXYB1C1Z), cytochrome P450 (CYP153; fdx-cyp-fdr) and alkB (alkB-rubA1-rubA2). Catabolic enzymes involved in ethene assimilation (EtnA, EtnC, EtnD, EtnE) and alkane assimilation (alcohol and aldehyde dehydrogenases) were identified by proteomics, and we showed for the first time that stress response proteins (catalase/peroxidase, chaperonins) were induced by growth on C2 -C5 alkanes and ethene. Surprisingly, none of the identified MO genes could be specifically associated with oxidation of small alkanes, and thus the nature of the gaseous alkane MO in NBB4 remains mysterious.

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH4) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH4 and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure–function relationships in the CuMMOs are inadequately understood. Here, we describe genetic and physiological characterization of a novel member of the CuMMO family that has an unusual physiological substrate range (C2–C4 alkanes) and a distinctive bacterial host (Mycobacterium). The Mycobacterial CuMMO genes (designated hmoCAB) were amenable to heterologous expression in M. smegmatis—this is the first example of recombinant expression of a complete and highly active CuMMO enzyme. The apparent specific activity of recombinant cells containing hmoCAB ranged from 2 to 3 nmol min–1 per mg protein on ethane, propane and butane as substrates, and the recombinants could also attack ethene, cis-dichloroethene and 1,2-dichloroethane. No detectable activity of recombinants or wild-type strains was seen with methane. The specific inhibitor allylthiourea strongly inhibited growth of wild-type cells on C2–C4 alkanes, and omission of copper from the medium had a similar effect, confirming the physiological role of the CuMMO for growth on alkanes. The hydrocarbon monooxygenase provides a new model for studying this important enzyme family, and the recombinant expression system will enable biochemical and molecular biological experiments (for example, site-directed mutagenesis) that were previously not possible.

Genome Sequence of the Ethene- and Vinyl Chloride-Oxidizing Actinomycete Nocardioides sp. Strain JS614

Nocardioides sp. strain JS614 grows on ethene and vinyl chloride (VC) as sole carbon and energy sources and is of interest for bioremediation and biocatalysis. Sequencing of the complete genome of JS614 provides insight into the genetic basis of alkene oxidation, supports ongoing research into the physiology and biochemistry of growth on ethene and VC, and provides biomarkers to facilitate detection of VC/ethene oxidizers in the environment. This is the first genome sequence from the genus Nocardioides and the first genome of a VC/ethene-oxidizing bacterium.

Unusual Class 1 Integron Configuration Found in Salmonella Genomic Island 2 from Salmonella enterica Serovar Emek

ABSTRACT Salmonella genomic island 2 (SGI2) is an independently derived genomic island related to SGI1 with the integron in a different position. The integron in SGI2 was found to include an additional 2.1 kb derived from the tni module of Tn5058, Tn502, or Tn512 that was not detected previously. Independent evolution of the backbone was confirmed with 21 single base differences found in over 11.5 kb, representing 40% of the 27.4-kb SGI2 backbone.

Unusual Class 1 Integron-Associated Gene Cassette Configuration Found in IncA/C Plasmids from Salmonella enterica

ABSTRACT IncA/C plasmids carrying an unusual cassette configuration in a class 1 integron and five further shared resistance genes, aacC4, aphA1, hph, sul2, and tetA(D) were found in Salmonella enterica serovars Senftenberg and Ohio. A deletion formed using a short region of homology in the 5′ conserved segment and the orfF cassette created an array with only part of orfF followed by the aadA2 cassette. The IncA/C plasmids were not recoverable by conjugation, but additional conjugative resistance plasmids were present in some strains.

Assessment of functional diversity and structure of phytate-hydrolysing bacterial community in #Lolium perenne# rhizosphere

Background and aimsPlant growth is frequently limited by the availability of inorganic phosphorus (P) in the soil. In most soils, a considerable amount of the soil P is bound to organic molecules. Of these, phytate is the most abundant identifiable organic P form, but is not readily available to plants. In contrast, microorganisms have been shown to degrade phytate with high efficiency. The current study aims to characterize the members of the phytate-hydrolysing bacterial community in rhizosphere, and the molecular and enzymatic ability of these bacteria to degrade phytate.Methods and resultsThe phytate-hydrolysing bacterial community was characterized from the rhizosphere of plants cultivated in the presence or absence of phytate supplementation. Major changes in the bacterial community structure were observed with both culture-dependent and -independent methods, which highlighted the predominance of Proteobacteria and Actinobacteria. Phytase activity was detected for a range of rhizobacterial isolates as well as the presence of, β-propeller phytases (BPP) for both isolates and directly in a soil sample.ConclusionA wide taxonomic range of functional phytate utilizers have been discovered, in soil bacterial taxa that were previously not well known for their ability to utilise phytate as P or C sources. This study provides new insights into microbial carbon and phosphorus cycling in soil.

Pathogenesis-related protein expression in the apoplast of wheat leaves protected against leaf rust following application of plant extracts.

Leaf rust (Puccinia triticina) is a major disease of wheat. We tested aqueous leaf extracts of Jacaranda mimosifolia (Bignoniaceae), Thevetia peruviana (Apocynaceae), and Calotropis procera (Apocynaceae) for their ability to protect wheat from leaf rust. Extracts from all three species inhibited P. triticina urediniospore germination in vitro. Plants sprayed with extracts before inoculation developed significantly lower levels of disease incidence (number of plants infected) than unsprayed, inoculated controls. Sprays combining 0.6% leaf extracts and 2 mM salicylic acid with the fungicide Amistar Xtra at 0.05% (azoxystrobin at 10 μg/liter + cyproconazole at 4 μg/liter) reduced disease incidence significantly more effectively than sprays of fungicide at 0.1% alone. Extracts of J. mimosifolia were most active, either alone (1.2%) or in lower doses (0.6%) in combination with 0.05% Amistar Xtra. Leaf extracts combined with fungicide strongly stimulated defense-related gene expression and the subsequent accumulation of pathogenesis-related (PR) proteins in the apoplast of inoculated wheat leaves. The level of protection afforded was significantly correlated with the ability of extracts to increase PR protein expression. We conclude that pretreatment of wheat leaves with spray formulations containing previously untested plant leaf extracts enhances protection against leaf rust provided by fungicide sprays, offering an alternative disease management strategy.

Seasonal total methane depletion in limestone caves

Methane concentration in caves is commonly much lower than the external atmosphere, yet the cave CH4 depletion causal mechanism is contested and dynamic links to external diurnal and seasonal temperature cycles unknown. Here, we report a continuous 3-year record of cave methane and other trace gases in Jenolan Caves, Australia which shows a seasonal cycle of extreme CH4 depletion, from ambient ~1,775 ppb to near zero during summer and to ~800 ppb in winter. Methanotrophic bacteria, some newly-discovered, rapidly consume methane on cave surfaces and in external karst soils with lifetimes in the cave of a few hours. Extreme bacterial selection due to the absence of alternate carbon sources for growth in the cave environment has resulted in an extremely high proportion 2–12% of methanotrophs in the total bacteria present. Unexpected seasonal bias in our cave CH4 depletion record is explained by a three-step process involving methanotrophy in aerobic karst soil above the cave, summer transport of soil-gas into the cave through epikarst, followed by further cave CH4 depletion. Disentangling cause and effect of cave gas variations by tracing sources and sinks has identified seasonal speleothem growth bias, with implied palaeo-climate record bias.

Heterologous Expression of Mycobacterium Alkene Monooxygenases in Gram-Positive and Gram-Negative Bacterial Hosts

Alkene MOs are of interest for their potential roles in industrial biocatalysis, most notably for the stereoselective synthesis of epoxides. Wild-type bacteria that grow on alkenes have high activities for alkene oxidation but are problematic for biocatalysis, since they tend to consume the epoxide products. Using recombinant biocatalysts is the obvious alternative, but a major bottleneck is the low activities of recombinant alkene MOs. Here, we provide new high-activity recombinant biocatalysts for alkene oxidation, and we provide insights into how to further improve these systems. ABSTRACT Alkene monooxygenases (MOs) are soluble di-iron-containing enzymes found in bacteria that grow on alkenes. Here, we report improved heterologous expression systems for the propene MO (PmoABCD) and ethene MO (EtnABCD) from Mycobacterium chubuense strain NBB4. Strong functional expression of PmoABCD and EtnABCD was achieved in Mycobacterium smegmatis mc2155, yielding epoxidation activities (62 and 27 nmol/min/mg protein, respectively) higher than any reported to date for heterologous expression of a di-iron MO system. Both PmoABCD and EtnABCD were specialized for the oxidation of gaseous alkenes (C2 to C4), and their activity was much lower on liquid alkenes (C5 to C8). Despite intensive efforts to express the complete EtnABCD enzyme in Escherichia coli, this was not achieved, although recombinant EtnB and EtnD proteins could be purified individually in soluble form. The biochemical function of EtnD as an oxidoreductase was confirmed (1.36 μmol cytochrome c reduced/min/mg protein). Cloning the EtnABCD gene cluster into Pseudomonas putida KT2440 yielded detectable epoxidation of ethene (0.5 nmol/min/mg protein), and this could be stimulated (up to 1.1 nmol/min/mg protein) by the coexpression of cpn60 chaperonins from either Mycobacterium spp. or E. coli. Successful expression of the ethene MO in a Gram-negative host was validated by both whole-cell activity assays and peptide mass spectrometry of induced proteins seen on SDS-PAGE gels. IMPORTANCE Alkene MOs are of interest for their potential roles in industrial biocatalysis, most notably for the stereoselective synthesis of epoxides. Wild-type bacteria that grow on alkenes have high activities for alkene oxidation but are problematic for biocatalysis, since they tend to consume the epoxide products. Using recombinant biocatalysts is the obvious alternative, but a major bottleneck is the low activities of recombinant alkene MOs. Here, we provide new high-activity recombinant biocatalysts for alkene oxidation, and we provide insights into how to further improve these systems.

Aqueous peat extract exposes rhizobia to sub-lethal stress which may prime cells for improved desiccation tolerance

Inoculation of legume seed with rhizobia is an efficient and cost-effective means of distributing elite rhizobial strains to broad-acre crops and pastures. However, necessary drying steps after coating seed expose rhizobia to desiccation stress reducing survival and limiting potential nitrogen fixation by legumes. Rhizobial tolerance to desiccation varies with strain and with growth conditions prior to drying. Cells grown in peat generally survive desiccation better than cells grown in liquid broth. We aimed to identify peat-induced proteomic changes in rhizobia that may be linked to desiccation tolerance. Proteins expressed differentially after growth in peat extract when compared with a minimal defined medium were measured in four rhizobial strains. Proteins showing the greatest increase in abundance were those involved in amino acid and carbohydrate transport and metabolism. Proteins involved in posttranslational modification and cell defence mechanisms were also upregulated. Many of the proteins identified in this study have been previously linked to stress responses. In addition, analysis using nucleic acid stains SYTO9 and propidium iodide indicated that membranes had been compromised after growth in peat extract. We targeted the membrane repair protein PspA (ΔRL3579) which was upregulated in Rhizobium leguminosarum bv. viceae 3841 after growth in peat extract to validate whether the inability to repair membrane damage after growth in peat extract reduced desiccation tolerance. The ΔRL3579 mutant grown in peat extract had significantly lower survival under desiccation stress, whereas no difference in survival between wild-type and mutant strains was observed after growth in tryptone yeast (TY) or minimal medium (JMM) media. Staining mutant and wild-type strains with SYTO9 and propidium iodide indicated that membranes of the mutant were compromised after growth in peat extract and to a lesser extent in TY. This study shows that growth in peat extract causes damage to cell membranes and exposes rhizobia to sub-lethal stress resulting in differential expression of several stress-induced proteins. The induction of these proteins may prime and protect the cells when subjected to subsequent stress such as desiccation. Identifying the key proteins involved in desiccation tolerance and properties of peat that stimulate this response will be important to inform development of new inoculant technology that maximises survival of rhizobia during delivery to legume crops and pastures.