Microbial Biotechnology

Published by Wiley Open Access
Online ISSN: 1751-7915
Print ISSN: 1751-7907
Classical medical research is disease focused and still defines health as absence of disease. Languages, however, associate a positive concept of wholeness with health as does the WHO health definition. Newer medical health definitions emphasize the capacity to adapt to changing external and internal circumstances. The results of the 2010 Global Burden of Disease study provides keys for a quantifiable health metrics by developing statistical tools calculating healthy life expectancy. Of central social and economic importance is the question whether healthy ageing can be achieved. This concept hinges on theories on the biological basis of lifespan determination and whether negligible senescence and the compression of morbidity can be achieved in human societies. Since the health impact of the human gut microbiome is currently a topical research area, microbiologists should be aware of the problems in defining health.
Average of specific OE activities of the LDS in Cx medium with EHCO, wheat bran or sucrose (control) as sole carbon and energy source at 24, 48, 72, 96 and 120 h of growth: (A) LACp, (B) LIPp and (C) MNPp. Dissimilar letters show significant differences (P ≤ 0.05). LACp, laccase; LIPp, lignin peroxidase; MNPp, manganese peroxidase.
Comparison of normalized second derivative ATR-FT-IR spectra of maltenes (A) and asphaltenes (B) fractions from biotreated EHCO and non-biotreated EHCO (control). Significant differences (P ≤ 0.05) among biotreatment and control are indicated for the OH (3460–3110 cm−1); C=O (1760–1650 cm−1); C=C (1651–1550 cm−1); SO (1303–930 cm−1); and OOP (915–707 cm−1) regions.
Growth rate of P. palmarum BM-04 in BSM medium with different concentrations of NaCl (A, C) and EHCO (B, D).
Growth percentage of P. palmarum BM-04 in BSM medium with different concentrations of NaCl (grey colour) and EHCO (black colour). Half maximal effective concentration to reduce the 50% of growth (EC50) is showed. Dissimilar letters show significant differences (P ≤ 0.05).
UV visible absorption spectra of asphaltenes fraction from biotreated EHCO (Asf.27) and non-biotreated EHCO (Asf.30).
Large amount of drilling waste associated with the expansion of the Orinoco Oil Belt (OOB), the biggest proven reserve of extra-heavy crude oil (EHCO) worldwide, is usually impregnated with EHCO and highly salinized water-based drilling fluids. Oxidative exoenzymes (OE) of the lignin-degrading enzyme system (LDS) of fungi catalyse the oxidation of a wide range of toxic pollutants. However, very little evidences on fungal degradation or biotransformation of EHCO have been reported, which contain high amounts of asphaltenes and its biodegradation rate is very limited. The aims of this work were to study the ability of Pestalotiopsis palmarum BM-04 to synthesize OE, its potential to biotransform EHCO and to survive in extreme environmental conditions. Enzymatic studies of the LDS showed the ability of this fungus to overproduce high amounts of laccase (LACp) in presence of wheat bran or lignin peroxidase (LIPp) with EHCO as sole carbon and energy source (1300 U mgP(-1) in both cases). FT-IR spectroscopy with Attenuated Total Reflectance (ATR) analysis showed the enzymatic oxidation of carbon and sulfur atoms in both maltenes and asphaltenes fractions of biotreated EHCO catalysed by cell-free laccase-enriched OE using wheat bran as inducer. UV-visible spectrophotometry analysis revealed the oxidation of the petroporphyrins in the asphaltenes fraction of biotreated EHCO. Tolerance assays showed the ability of this fungus to grow up to 50 000 p.p.m. of EHCO and 2000 mM of NaCl. These results suggest that P. palmarum BM-04 is a hopeful alternative to be used in remediation processes in extreme environmental conditions of salinity and EHCO contamination, such as the drilling waste from the OOB.
Pseudomonas sp. MX-058 produces aldehyde oxidase catalysing glyoxal to glyoxylic acid. Two aldehyde oxidases (F10 and F13) were purified to homogeneity from Pseudomonas sp. MX-058. F10 and F13 had subunit structures, a heterotetramer and heteropentamer respectively. The N-terminal amino acid sequences of all subunits were highly homologous to amino acid sequences of the putative oxidoreductases of Pseudomonas strains. All of these homologous oxidoreductases have a heterotrimer structure consisting of 85-88 (α), 37-39 (β) and 18-23 (γ) kDa subunits. However, the α-subunits of F10 and F13 might have decomposed into two [80 (α(1)) and 9 kDa (α(2))] and three [58 (α(1')), 22 (α(1″)) and 9 (α(2)) kDa] subunits, respectively, while the β- and γ-subunits remained intact. Both F10 and F13 show high activity toward several aliphatic and aromatic aldehydes. The aldehyde oxidases of Pseudomonas sp. MX-058 has unique protein structures, α(1)α(2)βγ for F10 and α(1')α(1″)α(2)βγ for F13, a heterotetramer and heteropentamer respectively. The enzymes exhibit significantly low activity toward glyoxylic acid compared with glyoxal, which is an advantageous property for glyoxylic acid production from glyoxal.
Comparison of the rRNA gene similarities of strain ES5, other Trichococcus strains. The bar represents 0.01% sequence difference.
Comparison of anaerobic substrate utilization by strain ES5 and Trichococcus strain R210 and Trichococcus flocculiformis DSM 2094 T .
Comparison of product formation by glycerol fermenting PDO-producing bacteria grown in batch culture. Data are given in molar percentages.
A coccal bacterium (strain ES5) was isolated from methanogenic bioreactor sludge with glycerol as the sole energy and carbon source. Strain ES5 fermented glycerol to 1,3-propanediol as main product, and lactate, acetate and formate as minor products. The strain was phylogenetically closely related to Trichococcus flocculiformis; the rRNA gene sequence similarity was 99%. However, strain ES5 does not show the typical growth in chains of T. flocculiformis. Moreover, T. flocculiformis does not ferment glycerol. Strain ES5 used a variety of sugars for growth. With these substrates, lactate, acetate and formate were the main products, while 1,3-propanediol was not formed. The optimum growth temperature of strain ES5 ranges from 30-37°C, but like several other Trichoccoccus strains, strain ES5 is able to grow at low temperature (< 10°C). Therefore, strain ES5 may be an appropriate catalyst for the biotechnological production of 1,3-propanediol from glycerol at low ambient temperature.
Streptomyces cinnamonensis DSM 1042 produces two types of isoprenoid secondary metabolites: the prenylated naphthalene derivative furanonaphthoquinone I (FNQ I), and isoprenylated phenazines which are termed endophenazines. Previously, a 55 kb gene cluster was identified which contained genes for both FNQ I and endophenazine biosynthesis. However, several genes required for the biosynthesis of these metabolites were not present in this cluster. We now re-screened the cosmid library for genes of the mevalonate pathway and identified a separate genomic locus which contains the previously missing genes. This locus (15 kb) comprised orthologues of four phenazine biosynthesis genes known from Pseudomonas strains. Furthermore, the locus contained a putative operon of six genes of the mevalonate pathway, as well as the gene epzP which showed sequence similarity to a recently discovered class of prenyltransferases. Inactivation and complementation experiments proved the involvement of epzP in the prenylation reaction in endophenazine biosynthesis. This newly identified genomic locus is more than 40 kb distant from the previously identified cluster. The protein EpzP was expressed in Escherichia coli in form of a his-tag fusion protein and purified. The enzyme catalysed the prenylation of 5,10-dihydrophenazine-1-carboxylic acid (dihydro-PCA) using dimethylallyl diphosphate (DMAPP) as isoprenoid substrate. K(m) values were determined as 108 µM for dihydro-PCA and 25 µM for DMAPP.
Venn diagrams of shared OTUs between the two samples (D07 and D08) for (A) 16S rRNA gene and (B) amoA gene. The number of reads that the OTUs represent is indicated in brackets.
Rarefaction curves of 16S rRNA OTUs defined by 3% sequence variations in the activated sludge. Curves refer to the pyrosequence analyses of the 2 consecutive years (D07 and D08-454) compared with the clone library analysis of the 2007 sludge (D07 – cloning).
Maximum-likelihood tree of amoA gene. The tree was determined using approximately 491 unambiguously aligned positions of nucleic acid amoA sequences. Each sequence from this study (in bold) is representative of clustered amoA sequences in the WWTP activated sludge with an identity of 94%. Reference sequences from GenBank database are indicated by their accession number if they correspond to uncultured organisms or by the strain name if they belong to amoA sequences from bacterial strains. The tree was constructed with RAxML (http://bioinformatics.oxfordjournals.org/content/22/21/2688.full) using the GTRGAMMA model and an alignment made with MUSCLE (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC390337/). The sequence of methane monooxygenase subunit A from Methylococcus capsulatus strain Bath served as outgroup (GenBank Accession No. YP_115248). The scale bar indicates substitutions per site.
The bacterial community composition of activated sludge from a wastewater treatment plant (Almería, Spain) with the particularity of using seawater was investigated by applying 454-pyrosequencing. The results showed that Deinococcus-Thermus, Proteobacteria, Chloroflexi and Bacteroidetes were the most abundant retrieved sequences, while other groups, such as Actinobacteria, Chlorobi, Deferribacteres, Firmicutes, Planctomycetes, Spirochaetes and Verrumicrobia were reported at lower proportions. Rarefaction analysis showed that very likely the diversity is higher than what could be described despite most of the unknown microorganisms probably correspond to rare diversity. Furthermore, the majority of taxa could not be classified at the genus level and likely represent novel members of these groups. Additionally, the nitrifiers in the sludge were characterized by pyrosequencing the amoA gene. In contrast, the nitrifying bacterial community, dominated by the genera Nitrosomonas, showed a low diversity and rarefaction curves exhibited saturation. These results suggest that only a few populations of low abundant but specialized bacteria are responsible for removal of ammonia in these saline wastewater systems.
pI of I. lacteus MnP and T. versicolor laccase detected in the supernatants of fungal cultures using a native IEF analyses.
Enzyme activities detected in the cultures of I. lacteus (A) and T. versicolor (B). Laccase activities were measured by the oxidation of ABTS (Matsumura et al., 1986). Manganese peroxidase was assayed with 2,6-dimethoxyphenol (DeJong et al., 1994). Enzyme activities were expressed in units of the enzyme activity (U; 1 unit oxidized 1 μmol of substrate per minute) per gram of a dry weight of fungal biomass present in the cultures. Laccase (circles), manganese peroxidase (squares), EE2-stimulated cultures (full symbols), control cultures (open symbols).
Laccase transcript levels detected in T. versicolor cultures by qPCR. Degenerated Cu1F and Cu2R primers (Luis et al., 2004) and T. versicolor-specific LaccTv-F and LaccTv-R primers (Hiscox et al., 2010) were used for laccase gene detection. A standard two-gene quantification strategy with PCR efficiency correction was then used and gene transcript levels were expressed as a ratio of the transcript levels detected in EE2-treated and untreated control cultures (Plačková et al., 2012).
MnP transcript levels detected in I. lacteus cultures by qPCR using the degenerated mnp-U and mnp-L primers (Nagai et al., 2007). A standard two-gene quantification strategy with PCR efficiency correction was then used and gene transcript levels were expressed as a ratio of the transcript levels detected in EE2-treated and untreated control cultures (Plačková et al., 2012).
In vitro degradation of EE2 by fungal mycelium and culture supernatants. 10 mg l−1 EE2 was incubated with a sample of homogenized fungal mycelium and culture liquid for 24 h at 28°C. Reaction mixtures contained 60 mM NaAc buffer (pH 5.0), 10 mg l−1 EE2, 2.5% (v/v) DMSO, and 0.4 ml of culture liquid/60 mg of homogenized mycelium in a final volume of 2 ml. A parallel set of samples were incubated in the presence of 1 mM MnSO4 and 0.5 mM H2O2. Culture liquids and mycelium inactivated by 100°C for 30 min served as controls. After the incubation samples were extracted with ethyl acetate and subjected to HPLC analyses.
Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase (MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17α-ethinyloestradiol (EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2 ± 6.7 U g(-1) of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene (lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T. versicolor, Irpex lacteus was also able to remove 10 mg l(-1) EE2 within 3 days of cultivation. While an increase to I. lacteus MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation.
Manganese (II) and manganese-oxidizing bacteria were used as an efficient biological system for the degradation of the xenoestrogen 17α-ethinylestradiol (EE2) at trace concentrations. Mn(2+)-derived higher oxidation states of Mn (Mn(3+), Mn(4+)) by Mn(2+)-oxidizing bacteria mediate the oxidative cleavage of the polycyclic target compound EE2. The presence of manganese (II) was found to be essential for the degradation of EE2 by Leptothrix discophora, Pseudomonas putida MB1, P. putida MB6 and P. putida MB29. Mn(2+)-dependent degradation of EE2 was found to be a slow process, which requires multi-fold excess of Mn(2+) and occurs in the late stationary phase of growth, implying a chemical process taking place. EE2-derived degradation products were shown to no longer exhibit undesirable estrogenic activity.
The marine organism Rhodopirellula baltica is a representative of the globally distributed phylum Planctomycetes whose members exhibit an intriguing lifestyle and cell morphology. The analysis of R. baltica's genome has revealed many biotechnologically promising features including a set of unique sulfatases and C1-metabolism genes. Salt resistance and the potential for adhesion in the adult phase of the cell cycle were observed during cultivation. To promote the understanding of this model organism and to specify the functions of potentially useful genes, gene expression throughout a growth curve was monitored using a whole genome microarray approach. Transcriptional profiling suggests that a large number of hypothetical proteins are active within the cell cycle and in the formation of the different cell morphologies. Numerous genes with potential biotechnological applications were found to be differentially regulated, revealing further characteristics of their functions and regulation mechanisms. More specifically, the experiments shed light on the expression patterns of genes belonging to the organism's general stress response, those involved in the reorganization of its genome and those effecting morphological changes. These transcriptomic results contribute to a better understanding of thus far unknown molecular elements of cell biology. Further, they pave the way for the biotechnological exploitation of R. baltica's distinctive metabolic features as a step towards sourcing the phylum Planctomycetes at large.
The reduction of quinones and humics by C. humireducens MFC-5 with sucrose as the electron donor: (a) AQDS; (b) AQS; (c) AQC; (d) FA; (e) HA. The experiments were performed under anaerobic conditions at 30°C for 10 days. Error bars represent standard deviation of the mean (n = 3).
Production of Fe(II) during the bioreduction of 5 mmol l−1 α-FeOOH by C. humireducens MFC-5 provided with 5 mmol l−1 sucrose as the electron donor and in the presence of electron transfer mediators (quinones or humics): (a) dissolved Fe(II); (b) total Fe(II). Inset shows total Fe(II) production from α-FeOOH reduction by MFC-5 with sucrose serving as the electron donor (without sucrose or active cells as control). The experiments were performed under anaerobic conditions at 30°C for 20 days. Error bars represent standard deviation of the mean (n = 3).
Linear correlation of total Fe(II) produced from α-FeOOH reduction with EAC of five types of electron shuttles in the α-FeOOH/MFC-5/humics (quinones)/sucrose system. Error bars represent standard deviation of the mean (n = 3).
The transformation rate of 2,4-D in the MFC-5/humics (quinones) system with sucrose as the electron donor. Three types of treatments served as controls: MFC-5 + 2,4-D + sucrose, MFC-5 + 2,4-D, 2,4-D + sucrose + dead cells. The experiments were performed under anaerobic conditions at 30°C for 16 days. Error bars represent standard deviation of the mean (n = 3).
Cyclic voltammograms of quinones, 2,4-D and 2,4-D/quinones under aqueous conditions. (a) AQDS; (b) AQS; (c) AQC. Working electrode, Pt disk (3 mm diameter); counter electrode, Hg wire; reference electrode, Ag/AgCl; 500 mmol l−1 NaClO4 electrolyte; scan rate, 50 mVs−1.
With the use of an alkaliphilic bacterium, Corynebacterium humireducens MFC-5, this study investigated the reduction of goethite (α-FeOOH) and degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) mediated by different humic substances (humics) and quinones in alkaline conditions (pH of 9.0). The results indicated that (i) using sucrose as the electron donor, the strain MFC-5 was capable of reducing anthraquinone-2,6-disulfonic acid (AQDS), anthraquinone-2-disulfonic acid (AQS), anthraquinone-2-carboxylic acid (AQC), humic acid (HA) and fulvic acid (FA), and its reducing capability ranked as AQC > AQS > AQDS > FA > HA; (ii) the anaerobic reduction of α-FeOOH and 2,4-D by the strain was insignificant, while the reductions were greatly enhanced by the addition of quinones/humics serving as redox mediators; (iii) the Fe(III) reduction rate was positively related to the content of quinone functional groups and the electron-accepting capacities (EAC) of quinones/humics based on fourier-transform infrared spectroscopy (FT-IR) and electrochemical analyses; however, such a relationship was not found in 2,4-D degradation probably because quinone reduction was not the rate-limiting step of quinone-mediated reduction of 2,4-D. Using the example of α-FeOOH and 2,4-D, this study well demonstrated the important role of humics reduction on the Fe(III)/Fe(II) biogeochemical cycle and chlorinated organic compounds degradation in alkaline reducing environments. Funding Information This study was supported by the National Natural Science Foundation of China (Nos 41101211, 31070460, 41101477), and The Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
A. UPGMA tree and heat map showing the phylogenetic relationships and abundance of the isolated strains for each of the different soils. The identified species correspond to the 68 clusters of different 16S rRNA gene ARDRA profiles, grouped based on 99% nucleotide similarity of the 16S rRNA gene sequences. From left to right: the UPGMA phylogenetic tree constructed with Geneious using the 16S rRNA gene sequences, heat map showing the abundance of the species in cfu g−1 soil for DS and GS obtained on 1/10 rich medium (yellow: 0 cfu g−1; light blue: 0.1–103 cfu g-1, blue: 103–105 cfu g−1, dark blue: 105–106 cfu g−1 and red: 106–107 cfu g−1) and the relative abundances in % of the species in each soil after enrichment cultures (EC) of the soils in the colour codes (yellow: 0; orange: 1–25%; dark-red (25–75%) and purple (75–100%). The corresponding strain number is shown next to the heat map, the most closely related bacterial 16S rRNA gene sequence and Genbank accession number of the most closely related species. B. Pie diagrams showing the bacteria family distribution of the different soils (in percentage %) on 1/10 rich medium (DS-s,w; GS-s,w) and isolated after successive dilution cultures with 2,4-DNT [enrichment culture 2,4-DNT contaminated soil (ECDS)-s,w; ECGS-s,w]. Calculations were performed on the different 16S rRNA gene identified strains.
A. Number of isolates grouped per genus, scoring positive for auxanography using 2,4-DNT as sole N-source. DS: 2,4-DNT contaminated soil summer and winter combined, GS: grassland soil summer and winter combined. B. Positive auxanography response towards 2,4-DNT as sole N-source by Ralstonia sp. HC90 (a) and negative auxanography response towards 2,4-DNT by Paenibacillus sp. HC2 (b). A sample of bacteria in MgSO4-buffer (100 μl of 107 cfu ml−1) was spread evenly over the whole surface of the minimal agar plate. After drying overnight, a drop of 2,4-DNT (30 μmol dissolved in DMSO) was spot on the plate and spread-out in half a circle, as shown in the scheme (c). After 5 days of incubation at 30°C, growth of bacteria was observed as white colonies. Ralstonia sp. HC90 grows only on the 2,4-DNT treated zone of the plate (+); Paenibacillus sp. HC2 strictly avoided growth on the 2,4-DNT treated zone (−).
Metabolization of 2,4-DNT by three consortia in liquid cultures under carbon limiting conditions (500 μM of 2,4-DNT was added as sole energy source). Y-axis: (left) concentration of the explosives in μM; (right) bacterial protein concentration (mg l−1) measured by the Bradford assay; X-axis: time in hours.A. Consortium UHS1 (Pseudomonas mandelii HC88, V. paradoxus VM685 and Burkholderia sp. HC114).B. Consortium UHS2 (V. paradoxus VM685; Burkholderia sp. HC114, Pseudomonas mandelii HC88, Ralstonia sp. HC90 and Burkholderia phytofirmans HC106).C. Consortium UHS3 (V. paradoxus VM685; Burkholderia sp. HC114, Pseudomonas mandelii HC88, Ralstonia sp. HC90, Burkholderia phytofirmans HC106, Bacillus subtilis HC45 and Variovorax sp. HC92).
A. Root length of A. thaliana seedlings non-exposed (bars in white) and exposed to 1 mg l−1 2,4-DNT for 9 days (bars in grey) in the absence or presence of consortia UHS1, UHS2, UHS3 (106 cfu ml−1). Values are mean ± standard error of at least 15 biological replicates. (Significance level versus control: *** P < 0.001, non-parametric Kruskal–Wallis test). B. Pictures of root hairs were taken under the binocular (20×) of (a) un-inoculated 2,4-DNT exposed plants and (b) 2,4-DNT exposed plants inoculated with UHS3.
The presence of explosives in soils and the interaction with drought stress and nutrient limitation are among the environmental factors that severely affect plant growth on military soils. In this study, we seek to isolate and identify the cultivable bacteria of a 2,4-dinitrotoluene (DNT) contaminated soil (DS) and an adjacent grassland soil (GS) of a military training area aiming to isolate new plant growth-promoting (PGP) and 2,4-DNT-degrading strains. Metabolic profiling revealed disturbances in Ecocarbon use in the bare DS; isolation of cultivable strains revealed a lower colony-forming-unit count and a less diverse community associated with DS in comparison with GS. New 2,4-DNT-tolerant strains were identified by selective enrichments, which were further characterized by auxanography for 2,4-DNT use, resistance to drought stress, cold, nutrient starvation and PGP features. By selecting multiple beneficial PGP and abiotic stress-resistant strains, efficient 2,4-DNT-degrading consortia were composed. After inoculation, consortium UHasselt Sofie 3 with seven members belonging to Burkholderia, Variovorax, Bacillus, Pseudomonas and Ralstonia species was capable to successfully enhance root length of Arabidopsis under 2,4-DNT stress. After 9 days, doubling of main root length was observed. Our results indicate that beneficial bacteria inhabiting a disturbed environment have the potential to improve plant growth and alleviate 2,4-DNT stress.
Pseudomonas putida KT2440 grows in M9 minimal medium with glucose in the presence of 2,4,6-trinitrotoluene (TNT) at a similar rate than in the absence of TNT, although global transcriptional analysis using DNA microarrays revealed that TNT exerts some stress. Response to TNT stress is regulated at the transcriptional level, as significant changes in the level of expression of 65 genes were observed. Of these genes, 39 appeared upregulated, and 26 were downregulated. The identity of upregulated genes suggests that P. putida uses two kinds of strategies to overcome TNT toxicity: (i) induction of genes encoding nitroreductases and detoxification-related enzymes (pnrA, xenD, acpD) and (ii) induction of multidrug efflux pump genes (mexEF/oprN) to reduce intracellular TNT concentrations. Mutants of 13 up- and 7 downregulated genes were analysed with regards to TNT toxicity revealing the role of the MexE/MexF/OprN pump and a putative isoquinoline 1-oxidoreductase in tolerance to TNT. The ORF PP1232 whose transcriptional level did not change in response to TNT affected growth in the presence of nitroaromatic compounds and it was found in a screening of 4000 randomly generated mutants.
pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy oriented two-step PCR cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300-based binary vector carrying a hygromycin resistance cassette, the pHg/pSILBAγ plasmid is used for Agrobacterium-mediated transformation. The pHg/pSILBAγ system results in predominantly single integrations of RNA silencing triggering T-DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. pSILBAγ construct and two other pSILBA plasmid variants (pSILBA and pSILBAα) were evaluated for their capacity to silence Laccaria nitrate reductase gene. While all pSILBA variants tested resulted in up to 65-76% of transformants with reduced growth on nitrate, pSILBAγ produced the highest number (65%) of strongly affected fungal strains. The strongly silenced phenotype was shown to correlate with T-DNA integration in transcriptionally active genomic sites. pHg/pSILBAγ was shown to produce T-DNAs with minimum CpG methylation in transgene promoter regions which assures the maximum silencing trigger production in Laccaria. Methylation of the target endogene was only slight in RNA silencing triggered with constructs carrying an intronic spacer hairpin sequence. The silencing capacity of the pHg/pSILBAγ was further tested with Laccaria inositol-1,4,5-triphosphate 5-phosphatase gene. Besides its use in silencing triggering, the herein described plasmid system can also be used for transgene expression in Laccaria. pHg/pSILBAγ silencing system is optimized for L. bicolor but it should be highly useful also for other homobasidiomycetes, group of fungi currently lacking molecular tools for RNA silencing.
In this feature, leading researchers in the field of environmental microbiology speculate on the technical and conceptual developments that will drive innovative research and open new vistas over the next few years.
The production of biofuels via microbial biotechnology is a very active field of research. A range of fuel molecule types are currently under consideration: alcohols, ethers, esters, isoprenes, alkenes and alkanes. At the present, the major alcohol biofuel is ethanol. The ethanol fermentation is an old technology. Ongoing efforts aim to increase yield and energy efficiency of ethanol production from biomass. n-Butanol, another microbial fermentation product, is potentially superior to ethanol as a fuel but suffers from low yield and unwanted side-products currently. In general, biodiesel fuels consist of fatty acid methyl esters in which the carbon derives from plants, not microbes. A new biodiesel product, called microdiesel, can be generated in engineered bacterial cells that condense ethanol with fatty acids. Perhaps the best fuel type to generate from biomass would be biohydrocarbons. Microbes are known to produce hydrocarbons such as isoprenes, long-chain alkenes and alkanes. The biochemical mechanisms of microbial hydrocarbon biosynthesis are currently under study. Hydrocarbons and minimally oxygenated molecules may also be produced by hybrid chemical and biological processes. A broad interest in novel fuel molecules is also driving the development of new bioinformatics tools to facilitate biofuels research.
The phnA gene that encodes the carbon-phosphorus bond cleavage enzyme phosphonoacetate hydrolase is widely distributed in the environment, suggesting that its phosphonate substrate may play a significant role in biogeochemical phosphorus cycling. Surprisingly, however, no biogenic origin for phosphonoacetate has yet been established. To facilitate the search for its natural source we have constructed a whole-cell phosphonoacetate biosensor. The gene encoding the LysR-type transcriptional activator PhnR, which controls expression of the phosphonoacetate degradative operon in Pseudomonas fluorescens 23F, was inserted in the broad-host-range promoter probe vector pPROBE-NT, together with the promoter region of the structural genes. Cells of Escherichia coli DH5α that contained the resultant construct, pPANT3, exhibited phosphonoacetate-dependent green fluorescent protein fluorescence in response to threshold concentrations of as little as 0.5 µM phosphonoacetate, some 100 times lower than the detection limit of currently available non-biological analytical methods; the pPANT3 biosensor construct in Pseudomonas putida KT2440 was less sensitive, although with shorter response times. From a range of other phosphonates and phosphonoacetate analogues tested, only phosphonoacetaldehyde and arsonoacetate induced green fluorescent protein fluorescence in the E. coli DH5α (pPANT3) biosensor, although at much-reduced sensitivities (50 µM phosphonoacetaldehyde and 500 µM arsonoacetate).
The increasing availability of the genome sequences of microorganisms involved in important bioremediation processes makes it feasible to consider developing genome-scale models that can aid in predicting the likely outcome of potential subsurface bioremediation strategies. Previous studies of the in situ bioremediation of uranium-contaminated groundwater have demonstrated that Geobacter species are often the dominant members of the groundwater community during active bioremediation and the primary organisms catalysing U(VI) reduction. Therefore, a genome-scale, constraint-based model of the metabolism of Geobacter sulfurreducens was coupled with the reactive transport model HYDROGEOCHEM in an attempt to model in situ uranium bioremediation. In order to simplify the modelling, the influence of only three growth factors was considered: acetate, the electron donor added to stimulate U(VI) reduction; Fe(III), the electron acceptor primarily supporting growth of Geobacter; and ammonium, a key nutrient. The constraint-based model predicted that growth yields of Geobacter varied significantly based on the availability of these three growth factors and that there are minimum thresholds of acetate and Fe(III) below which growth and activity are not possible. This contrasts with typical, empirical microbial models that assume fixed growth yields and the possibility for complete metabolism of the substrates. The coupled genome-scale and reactive transport model predicted acetate concentrations and U(VI) reduction rates in a field trial of in situ uranium bioremediation that were comparable to the predictions of a calibrated conventional model, but without the need for empirical calibration, other than specifying the initial biomass of Geobacter. These results suggest that coupling genome-scale metabolic models with reactive transport models may be a good approach to developing models that can be truly predictive, without empirical calibration, for evaluating the probable response of subsurface microorganisms to possible bioremediation approaches prior to implementation.
Lantibiotics are antimicrobial peptides which contain a high percentage of post-translationally modified residues. While most attention has been paid to the role of these critical structural features, evidence continues to emerge that charged amino acids also play a key role in these peptides. Here 16 'charge' mutants of the two-peptide lantibiotic lacticin 3147 [composed of Ltnα (2+, 2-) and Ltnβ (2+)] were constructed which, when supplemented with previously generated peptides, results in a total bank of 23 derivatives altered in one or more charged residues. When examined individually, in combination with a wild-type partner or, in some instances, in combination with one another, these mutants reveal the importance of charge at specific locations within Ltnα and Ltnβ, confirm the critical role of the negatively charged glutamate residue in Ltnα and facilitate an investigation of the contribution of positively charged residues to the cationic Ltnβ. From these investigations it is also apparent that the relative importance of the overall charge of lacticin 3147 varies depending on the target bacteria and is most evident when strains with more negatively charged cell envelopes are targeted. These studies also result in, for the first time, the creation of a derivative of a lacticin 3147 peptide (LtnβR27A) which displays enhanced specific activity.
The lantibiotic lacticin 3147 consists of two ribosomally synthesized and post-translationally modified antimicrobial peptides, Ltnα and Ltnβ, which act synergistically against a wide range of Gram-positive microorganisms. We performed saturation mutagenesis of specific residues of Ltnα to determine their functional importance. The results establish that Ltnα is more tolerant to change than previously suggested by alanine scanning mutagenesis. One substitution, LtnαH23S, was identified which improved the specific activity of lacticin 3147 against one pathogenic strain, Staphylococcus aureus NCDO1499. This represents the first occasion upon which the activity of a two peptide lantibiotic has been enhanced through bioengineering. Funding Information Work in the authors' laboratory is supported by the Irish Government under the National Development Plan; by the Irish Research Council for Science Engineering and Technology (IRCSET); by Enterprise Ireland; and by Science Foundation Ireland (SFI), through the Alimentary Pharmabiotic Centre (APC) at University College Cork, Ireland, which is supported by the SFI-funded Centre for Science, Engineering and Technology (SFI-CSET) and provided P.D.C., C.H and R.P.R. with SFI Principal Investigator funding.
Olive knot disease, caused by Pseudomonas savastanoi pv. savastanoi, is one of the most important biotic constraints for olive cultivation. Pseudomonas fluorescens PICF7, a natural colonizer of olive roots and effective biological control agent (BCA) against Verticillium wilt of olive, was examined as potential BCA against olive knot disease. Bioassays using in vitro-propagated olive plants were carried out to assess whether strain PICF7 controlled knot development either when co-inoculated with the pathogen in stems or when the BCA (in roots) and the pathogen (in stems) were spatially separated. Results showed that PICF7 was able to establish and persist in stem tissues upon artificial inoculation. While PICF7 was not able to suppress disease development, its presence transiently decreased pathogen population size, produced less necrotic tumours, and sharply altered the localization of the pathogen in the hyperplasic tissue, which may pose epidemiological consequences. Confocal laser scanning microscopy combined with fluorescent tagging of bacteria revealed that when PICF7 was absent the pathogen tended to be localized at the knot surface. However, presence of the BCA seemed to confine P. savastanoi at inner regions of the tumours. This approach has also enabled to prove that the pathogen can moved systemically beyond the hypertrophied tissue.
'Candidatus Accumulibacter' is a biotechnologically important bacterial group that can accumulate large amounts of intracellular polyphosphate, contributing to biological phosphorus removal in wastewater treatment. Since its first molecular identification more than a decade ago, this bacterial group has drawn significant research attention due to its high abundance in many biological phosphorus removal systems. In the past 6 years, our understanding of Accumulibacter microbiology and ecophysiology has advanced rapidly, largely owing to genomic information obtained through shotgun metagenomic sequencing efforts. In this review, we focus on the metabolism, physiology, fine-scale population structure and ecological distribution of Accumulibacter, aiming to integrate the information learned so far and to present a more complete picture of the microbiology of this important bacterial group.
Accumulated concentration of extracellular pyruvate and alpha-ketoglutarate in batch-grown cultures of S6803 ΔglgC with limited nitrate in the inoculum. Cells were grown to an OD730 of approximately 1.0 in BG-11 (containing 17.6 mM NaNO3) under 50 μE* m−2 s−1 illumination from cool white fluorescent bulbs. These precultures were inoculated into BG-11 media with the indicated nitrate content and cultivated under the same illumination. Cultures were sampled regularly to measure (A) cell density (as OD730), (B) extracellular pyruvate and (C) extracellular alpha-ketoglutarate.
Whole-cell spectra of nitrate-free cultures of S6803 ΔglgC (A) over 9 days of photo-catalysis and (B) upon re-addition of 2 mM NaNO3 following 6 days of photo-catalysis. Cultures grown for 6 days in nitrate-limited medium in A are the same as those in B, labelled 0 days after nitrogen re-addition. Illumination was supplied by cool white fluorescent bulbs at an intensity of 50 μE* m−2 s−1.
Regeneration of photo-catalytic production of pyruvate and alpha-ketoglutarate from nitrate-free cell suspensions of S6803 ΔglgC by addition of 2 mM NaNO3 after 3 or 4 days of photo-catalysis. Illumination was supplied by cool white fluorescent bulbs at an intensity of 50 μE* m−2 s−1. Cells were grown to an OD730 of approximately 1.0 in BG-11 (containing 17.6 mM NaNO3) under 50 μE* m−2 s−1 from cool white fluorescent bulbs and re-suspended after washing in nitrate-free BG-11 to an OD730 of approximately 0.6. Cultures were sampled regularly to measure (A) cell density (as OD730), (B) extracellular pyruvate and (C) extracellular alpha-ketoglutarate.
Effect of continuous illumination intensity on photo-catalytic production of (A) pyruvate and (B) alpha-ketoglutarate, and (C) the calculated molar ratio of alpha-ketoglutarate/pyruvate yield. Cultures of S6803 ΔglgC were grown under 24 h of illumination from cool white LEDs at the indicated light intensities, harvested and washed in nitrate-free BG-11, and re-suspended at a cell density of 3.1 OD730. The new suspensions were returned to the same conditions in which they were grown and sampled daily to measure extracellular metabolites. The molar ratio of products from cultures illuminated at 50 μE* m−2s−1 had high uncertainty (because of detection limits of high-performance liquid chromatography for pyruvate) prior to 1 day after nitrogen removal and is therefore not shown in panel C.
A key objective in microbial biofuels strain development is to maximize carbon flux to target products while minimizing cell biomass accumulation, such that ideally the algae and bacteria would operate in a photo-catalytic state. A brief period of such a physiological state has recently been demonstrated in the cyanobacterium Synechocystis sp. PCC 6803 ΔglgC strain incapable of glycogen storage. When deprived of nitrogen, the ΔglgC excretes the organic acids alpha-ketoglutarate and pyruvate for a number of days without increasing cell biomass. This study examines the relationship between the growth state and the photo-catalytic state, and characterizes the metabolic adaptability of the photo-catalytic state to increasing light intensity. It is found that the culture can transition naturally from the growth state into the photo-catalytic state when provided with limited nitrogen supply during the growth phase. Photosynthetic capacity and pigments are lost over time in the photo-catalytic state. Reversal to growth state is observed with re-addition of nitrogen nutrient, accompanied by restoration of photosynthetic capacity and pigment levels in the cells. While the overall productivity increased under high light conditions, the ratio of alpha-ketoglutarate/pyruvate is altered, suggesting that carbon partition between the two products is adaptable to environmental conditions. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
Possible anaerobic benzene activation steps and further transformation reactions to benzoyl-CoA as central metabolite (modified from Foght, 2008 and Laban et al., 2010). (a) Methylation; (b) hydroxylation; (c) carboxylation. Benzoyl-CoA can be further reduced by ATP-dependent or -independent benzoyl-CoA reductases (Kung et al., 2009).
Benzene is a widespread and toxic contaminant. The fate of benzene in contaminated aquifers seems to be primarily controlled by the abundance of oxygen: benzene is aerobically degraded at high rates by ubiquitous microorganisms, and the oxygen-dependent pathways for its breakdown were elucidated more than 50 years ago. In contrast, benzene was thought to be persistent under anoxic conditions until 25 years ago. Nevertheless, within the last 15 years, several benzene-degrading cultures have been enriched under varying electron acceptor conditions in laboratories around the world, and organisms involved in anaerobic benzene degradation have been identified, indicating that anaerobic benzene degradation is a relevant environmental process. However, only a few benzene degraders have been isolated in pure culture so far, and they all use nitrate as an electron acceptor. In some highly enriched strictly anaerobic cultures, benzene has been described to be mineralized cooperatively by two or more different organisms. Despite great efforts, the biochemical mechanism by which the aromatic ring of benzene is activated in the absence of oxygen is still not fully elucidated; methylation, hydroxylation and carboxylation are discussed as likely reactions. This review summarizes the current knowledge about the 'key players' of anaerobic benzene degradation under different electron acceptor conditions and the possible pathway(s) of anaerobic benzene degradation.
Ferrioxamines-mediated iron acquisition by Streptomyces coelicolor A3(2) has recently received increased attention. In addition to the biological role of desferrioxamines (dFOs) as hydroxamate siderophores, and the pharmaceutical application of dFO-B as an iron-chelator, the ferrioxamines have been shown to mediate microbial interactions. In S. coelicolor the siderophore-binding receptors DesE (Sco2780) and CdtB (Sco7399) have been postulated to specifically recognize and uptake FO-E (cyclic) and FO-B (linear) respectively. Here, disruption of the desE gene in S. coelicolor, and subsequent phenotypic analysis, is used to demonstrate a link between iron metabolism and physiological and morphological development. Streptomyces coelicolor desE mutants, isolated in both wild-type (M145) and a coelichelin biosynthesis and transport minus background (mutant W3), a second hydroxamate siderophore system only found in S. coelicolor and related species, resulted in impaired growth and lack of sporulation. This phenotype could only be partially rescued by expression in trans of either desE and cdtB genes, which contrasted with the ability of FO-E, and to a lesser extent of FO-B, to fully restore growth at µM concentrations, with a concomitant induction of a marked phenotypic response involving precocious synthesis of actinorhodin and sporulation. Moreover, growth restoration of the desE mutant by complementation with desE and cdtB showed that DesE, which is universally conserved in Streptomyces, and CdtB, only present in certain streptomycetes, have partial equivalent functional roles under laboratory conditions, implying overlapping ferrioxamine specificities. The biotechnological and ecological implications of these observations are discussed.
Streptomycetes have high biotechnological relevance as producers of diverse metabolites widely used in medical and agricultural applications. The biosynthesis of these metabolites is controlled by signalling molecules, γ-butyrolactones, that act as bacterial hormones. In Streptomyces coelicolor, a group of signalling molecules called SCBs (S. coelicolorbutanolides) regulates production of the pigmented antibiotics coelicolor polyketide (CPK), actinorhodin and undecylprodigiosin. The γ-butyrolactone synthase ScbA is responsible for the biosynthesis of SCBs. Here we show the results of a genome-wide transcriptome analysis of a scbA deletion mutant prior to and during the transition to antibiotic production. We report a strong perturbation in the expression of three pigmented antibiotic clusters in the mutant throughout the growth curve, thus providing a molecular explanation for the antibiotic phenotype observed previously. Our study also revealed, for the first time, that the secondary metabolite cluster responsible for synthesis of the siderophore desferrioxamine is under the control of SCB signalling. Moreover, expression of the genes encoding enzymes for primary metabolism pathways, which supply antibiotic precursors and genes for morphological differentiation, was found shifted earlier in time in the mutant. In conclusion, our time series analysis demonstrates new details of the regulatory effects of the γ-butyrolactone system in Streptomyces.
TEM micrograph of PS/AA-ALN8T NPs (Inset is the photograph of the blue latex).
Zeta potential values of (a) p-Ab-PS/AA-ALN8T, (b) c-Ab-PS/AA-ALN8T and (c) aff-Ab-PS/AA-ALN8T, prepared at different ratios of Ab to the latex measured at various pHs (25°C, 1 mM NaCl).
OM images of the immunoagglutination of (row 1) p-Ab-PS/AA-ALN8T, (row 2) c-Ab-PS/AA-ALN8T, and (row 3) aff-Ab-PS/AA-ALN8T (5.0 μl) in the presence of (a) PBS, (b) malaria naive plasma, (c) P. falciparum-infected plasma and (d) P. vivax-infected plasma (0.5 μl) (mixing time 2 min).
Polystyrene (PS) nanoparticle (NP) copolymerized with acrylic acid (AA) and coloured monomer, i.e. 2,3,6,7-tetra(2,2'-bithiophene)-1,4,5,8-naphthalenetetracarboxylic-N,N'-di(2-methylallyl)-bisimide (ALN8T), was synthesized via the miniemulsion polymerization. Before applying for malaria antigen detection, the blue NP was conjugated with human polyclonal malaria IgG antibody (Ab) specific to Plasmodium falciparum. For the conjugation, three methods, i.e. physical adsorption, covalent coupling and affinity binding via streptavidin (SA) and biotin interaction, were employed. The optimum ratio of Ab to NPs used in each immobilization procedure and the latex agglutination test based on the reaction between Ab conjugated NPs and malaria patient plasma were investigated. All Ab-latex conjugates provided the high sensitivity for the detection of P. falciparum malaria plasma. The highest specificity to P. falciparum was obtained from using Ab-NPs conjugated via the SA-biotin interaction.
This study presents seasonal and spatial variations of the ammonifying bacteria (AB) and denitrifying bacteria (DNB) and physicochemical parameters in 10 lakes and reservoirs in the northeast of China. Water samples were collected in winter (January), spring (March), summer (July) and fall (November) in 2011. The study revealed that physicochemical parameters such as pH, dissolved oxygen (DO), NH4 (+) -N and nitrate as nitrogen were closely related with the distribution of AB and DNB. Seasonally, the levels of AB presents gradually upward trend from winter to summer, and declines in fall and DNB were higher in spring and fall than summer and lowest in winter. Spatially, the annual average of AB among 10 lakes and reservoirs showed insignificant difference (P > 0.05), for DNB, Udalianchi and Lianhuan Lake were lower than others (P < 0.05). Regression correlation analysis showed that the levels of AB and DNB had a close relationship with nitrogen nutrition. Three principal components were identified of total variances which are conditionally classified by the 'natural' factor (PC1) and 'nitrogen nutrients' (PC2, PC3). According the principal component scores, cluster analysis detected two distinct groups: (C1) mainly affected by nitrogen nutrients and (C2) natural environmental factors. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
Concerns have been raised about potential horizontal gene transfer (HGT) of antibiotic resistance markers (ARMs) from transgenic plants to bacteria of medical and environmental importance. All ARMs used in transgenic plants have been bacterial in origin, but it has been recently shown that an Arabidopsis thaliana ABC transporter, Atwbc19, confers kanamycin resistance when overexpressed in transgenic plants. Atwbc19 was evaluated for its ability to transfer kanamycin resistance to Escherichia coli, a kanamycin-sensitive model bacterium, under simulated HGT, staged by subcloning Atwbc19 under the control of a bacterial promoter, genetically transforming to kanamycin-sensitive bacteria, and assessing if resistance was conferred as compared with bacteria harbouring nptII, the standard kanamycin resistance gene used to produce transgenic plants. NptII provided much greater resistance than Atwbc19 and was significantly different from the no-plasmid control at low concentrations. Atwbc19 was not significantly different from the no-plasmid control at higher concentrations. Even though HGT risks are considered low with nptII, Atwbc19 should have even lower risks, as its encoded protein is possibly mistargeted in bacteria.
Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k(cat)/K(m))](CE)/[(k(cat)/K(m))](FAE) factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys(281), Asp(282), Asn(316) and Lys(317)) situated close to the catalytic core (Ser(143)/Asp(273)/His(305)) and a deletion of a 34-AA-long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k(cat)/K(m) values) and enzymes with inverted specificity ((k(cat)/K(m))(CE)/(k(cat)/K(m))(FAE) ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol(-1)), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol(-1)) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of 'hot spot' mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.
To find links between the biotic characteristics and abiotic process parameters in anaerobic digestion systems, the microbial communities of nine full-scale biogas plants in South Tyrol (Italy) and Vorarlberg (Austria) were investigated using molecular techniques and the physical and chemical properties were monitored. DNA from sludge samples was subjected to microarray hybridization with the ANAEROCHIP microarray and results indicated that sludge samples grouped into two main clusters, dominated either by Methanosarcina or by Methanosaeta, both aceticlastic methanogens. Hydrogenotrophic methanogens were hardly detected or if detected, gave low hybridization signals. Results obtained using denaturing gradient gel electrophoresis (DGGE) supported the findings of microarray hybridization. Real-time PCR targeting Methanosarcina and Methanosaeta was conducted to provide quantitative data on the dominating methanogens. Correlation analysis to determine any links between the microbial communities found by microarray analysis, and the physicochemical parameters investigated was conducted. It was shown that the sludge samples dominated by the genus Methanosarcina were positively correlated with higher concentrations of acetate, whereas sludge samples dominated by representatives of the genus Methanosaeta had lower acetate concentrations. No other correlations between biotic characteristics and abiotic parameters were found. Methanogenic communities in each reactor were highly stable and resilient over the whole year.
Sources of wild-type bacteria isolates and bacteriophages. 
Summary of real-time PCR diagnostics using Podoviridae and Myoviridae specific probe and primers. 
Summary of screening results for the presence of lysogeny in wild-type global populations of E. amylovora and P. agglomerans using real-time PCR. 
Lytic bacteriophages are in development as biological control agents for the prevention of fire blight disease caused by Erwinia amylovora. Temperate phages should be excluded as biologicals since lysogeny produces the dual risks of host resistance to phage attack and the transduction of virulence determinants between bacteria. The extent of lysogeny was estimated in wild populations of E. amylovora and Pantoea agglomerans with real-time polymerase chain reaction primers developed to detect E. amylovora phages belonging to the Myoviridae and Podoviridae families. Pantoea agglomerans, an orchard epiphyte, is easily infected by Erwinia spp. phages, and it serves as a carrier in the development of the phage-mediated biological control agent. Screening of 161 E. amylovora isolates from 16 distinct geographical areas in North America, Europe, North Africa and New Zealand and 82 P. agglomerans isolates from southern Ontario, Canada showed that none possessed prophage. Unstable phage resistant clones or lysogens were produced under laboratory conditions. Additionally, a stable lysogen was recovered from infection of bacterial isolate Ea110R with Podoviridae phage ΦEa35-20. These laboratory observations suggested that while lysogeny is possible in E. amylovora, it is rare or absent in natural populations, and there is a minimal risk associated with lysogenic conversion and transduction by Erwinia spp. phages. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
Specific methanogenic activity (SMA) against H2/CO2 (ml CH4 g−1 volatile suspended solids [VSS] -h−1) for each anaerobic enrichment culture (n = 13). SMA values from R1 and R3 were different (P < 0.05) from those from R2 and R4. Measurements not different from each other are indicated with the letters a or b.
qPCR data.A. Quantification of mcrA gene copies ng−1 DNA from multiple nucleic acid extractions of bioreactor biomass.B. Quantification of mcrA transcripts ng−1 RNA from the same samples of bioreactor biomass. Bars in both panels show standard error of the mean.
Relationship between mcrA gene copy ng−1 DNA abundance in the four bioreactors and SMA. qPCR results versus SMA were significant (r2 = 0.9779, P = 0.0074).
A. Methanogen genus assignments for mcrA clones. Relative abundance of mcrA clones in each library to specific methanogen genera based on 88% sequence similarity according to Steinberg and Regan (2008). B. Dendrogram using the Sorenson index representing the relationships among the three clone libraries from each enrichment culture. Grey branches label clone libraries from cultures with lower SMAs while black branches represent libraries from cultures with higher SMAs. The dendrogram also uses 88% sequence similarity to group sequences and so is based on genus relationships not species.
Biologically produced methane (CH4 ) from anaerobic digesters is a renewable alternative to fossil fuels, but digester failure can be a serious problem. Monitoring the microbial community within the digester could provide valuable information about process stability because this technology is dependent upon the metabolic processes of microorganisms. A healthy methanogenic community is critical for digester function and CH4 production. Methanogens can be surveyed and monitored using genes and transcripts of mcrA, which encodes the α subunit of methyl coenzyme M reductase - the enzyme that catalyses the final step in methanogenesis. Using clone libraries and quantitative polymerase chain reaction, we compared the diversity and abundance of mcrA genes and transcripts in four different methanogenic hydrogen/CO2 enrichment cultures to function, as measured by specific methanogenic activity (SMA) assays using H2 /CO2 . The mcrA gene copy number significantly correlated with CH4 production rates using H2 /CO2 , while correlations between mcrA transcript number and SMA were not significant. The DNA and cDNA clone libraries from all enrichments were distinctive but community diversity also did not correlate with SMA. Although hydrogenotrophic methanogens dominated these enrichments, the results indicate that this methodology should be applicable to monitoring other methanogenic communities in anaerobic digesters. Ultimately, this could lead to the engineering of digester microbial communities to produce more CH4 for use as renewable fuel.
Growth of C. glutamicum strains in CgXII medium containing 100 mM xylose.A. Corynebacterium glutamicum strains WT(pEKEx3-xylAXc) (open diamonds), WT(pEKEx3-xylABs) (open triangles), WT(pEKEx3-xylAEc) (closed circles) and WT(pEKEx3-xylAMs) (closed squares) were analysed.B. Corynebacterium glutamicum strains WT(pEKEx3-xylAXc-xylBBs) (open diamonds), WT(pEKEx3-xylAXc-xylBEc) (open triangles), WT(pEKEx3-xylAXc) (closed circles) and WT(pEKEx3-xylAXc-xylBCg) (closed squares) were analysed. Data represents mean values and standard deviations of three independent cultivations.
Product concentrations (A) and volumetric productivities (B) for l-glutamate, l-lysine, l-ornithine and putrescine production in CgXII medium containing 100 mM xylose. Corynebacterium glutamicum strains with pEKEx3-xylAEc or pEKEx3-xylAXc-xylBCg were analysed. l-glutamate was produced with WT (hatched bars), l-lysine with DM1729 (open bars), l-ornithine with ORN1 (closed bars) and putrescine with PUT21 (checked bars). Data represent mean values and experimental imprecision of two independent cultivations.
Product concentrations (A, C) and volumetric productivities (B, D) for l-glutamate (A, B) and l-lysine (C, D) production in CgXII medium containing rice straw hydrolysate. Corynebacterium glutamicum strains with empty vectors, pVWEx1-araBAD and pEKEx3-xylAEc or pVWEx1-araBAD and pEKEx3-xylAXc-xylBCg were analysed. l-glutamate was produced with WT (hatched bars) and l-lysine with DM1729 (open bars). Data represent mean values and experimental imprecision of two independent cultivations.
Because of their abundance in hemicellulosic wastes arabinose and xylose are an interesting source of carbon for biotechnological production processes. Previous studies have engineered several Corynebacterium glutamicum strains for the utilization of arabinose and xylose, however, with inefficient xylose utilization capabilities. To improve xylose utilization, different xylose isomerase genes were tested in C. glutamicum. The gene originating from Xanthomonas campestris was shown to have the highest effect, resulting in growth rates of 0.14 h−1, followed by genes from Bacillus subtilis, Mycobacterium smegmatis and Escherichia coli. To further increase xylose utilization different xylulokinase genes were expressed combined with X. campestris xylose isomerase gene. All combinations further increased growth rates of the recombinant strains up to 0.20 h−1 and moreover increased biomass yields. The gene combination of X. campestris xylose isomerase and C. glutamicum xylulokinase was the fastest growing on xylose and compared with the previously described strain solely expressing E. coli xylose isomerase gene delivered a doubled growth rate. Productivity of the amino acids glutamate, lysine and ornithine, as well as the diamine putrescine was increased as well as final titres except for lysine where titres remained unchanged. Also productivity in medium containing rice straw hydrolysate as carbon source was increased. Funding Information No funding information provided.
The current knowledge of trehalose biosynthesis under stress conditions is incomplete and needs further research. Since trehalose finds industrial and pharmaceutical applications, enhanced accumulation of trehalose in bacteria seems advantageous for commercial production. Moreover, physiological role of trehalose is a key to generate stress resistant bacteria by metabolic engineering. Although trehalose biosynthesis requires few metabolites and enzyme reactions, it appears to have a more complex metabolic regulation. Trehalose biosynthesis in bacteria is known through three pathways - OtsAB, TreYZ and TreS. The interconnections of in vivo synthesis of trehalose, glycogen or maltose were most interesting to investigate in recent years. Further, enzymes at different nodes (glucose-6-P, glucose-1-P and NDP-glucose) of metabolic pathways influence enhancement of trehalose accumulation. Most of the study of trehalose biosynthesis was explored in medically significant Mycobacterium, research model Escherichia coli, industrially applicable Corynebacterium and food and probiotic interest Propionibacterium freudenreichii. Therefore, the present review dealt with the trehalose metabolism in these bacteria. In addition, an effort was made to recognize how enzymes at different nodes of metabolic pathway can influence trehalose accumulation.
Osmotolerance of evolved strain IMZ333 (evolved Gpd–), ancestral strain IMZ160 (unevolved Gpd–) and the reference strain IME076 (Gpd+). Spot assay experiments were performed on synthetic medium agar plates with 0.1–1.0 M glucose under aerobic and anaerobic conditions. Pictures were taken after 3 days (A) and 7 days (B) of incubation at 30°C.
Anaerobic batch cultivation of the evolved osmotolerant strain S. cerevisiae IMZ333 (evolved Gpd–, A and C) and the reference strain IME076 (Gpd+, B) on synthetic medium with 1 M glucose. Both strains were grown at pH 5.0 and at 30°C.A. IMZ333, 2 g l−1 acetic acid.B. IME076, 2 g l−1 acetic acid.C. IMZ333, 3 g l−1 acetic acid. Symbols: ▲, Dry weight; •, glucose; ○, ethanol (not corrected for evaporation); ▪, acetate; □, glycerol. Each graph represents values for one of two independent replicates, which differ less than 5% in growth kinetics.
Analysis of the contributions of genomic and/or plasmid based mutations to the evolved osmotolerant phenotype of Gpd–S. cerevisiae. Aerobic (black bars) and anaerobic (grey-bars) shake flask cultures were both incubated at 30°C and at 200 r.p.m. with an initial glucose concentration of 1 M. The optical density (OD 660 nm) was measured after 48 h for strains IME076 (Gpd+ with empty-vector p426_GPD) and IMZ333 (evolved Gpd– with evolved pUDE043 population) or after 72 h for strains IMJ004 (evolved Gpd– pUDE043), IMJ005 (evolved Gpd– and pUDE043ev1), IMJ006 (evolved Gpd– and pUDE043ev2) and IMJ009 (evolved Gpd– with empty-vector p426_GPD).
Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd– mhpF-expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h−1. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l−1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth.
A microbial fuel cell (MFC) was operated with a pure culture of Cupriavidus basilensis bacterial cells growing in the anode compartment in a defined medium containing acetate or phenol. Operating this mediator-less MFC under a constant external resistor of 1 kΩ with acetate or phenol led to current generation of 902 and 310 mA m(-2) respectively. In the MFC which was operated using acetate or phenol, the current density measured from the plankton bacterial cells with a fresh electrode was 125 and 109 mA m(-2) , respectively, whereas the current obtained with biofilm-covered electrodes in sterile medium was 541 and 228 mA m(-2) respectively. After 72 h in the MFC, 86% of the initial phenol concentration was removed, while only 64% was removed after the same time in the control MFC which was held at an open circuit potential (OCP). Furthermore, SEM and confocal microscopy analyses demonstrated a developed biofilm with a live C. basilensis population. In conclusion, in this study we demonstrated, for the first time, use of C. basilensis facultative aerobe bacterial cells in a MFC using acetate or phenol as the sole carbon source which led to electricity generation.
In a continuous culture under phosphate limitation the metabolism of Clostridium acetobutylicum depends on the external pH level. By comparing seven steady-state conditions between pH 5.7 and pH 4.5 we show that the switch from acidogenesis to solventogenesis occurs between pH 5.3 and pH 5.0 with an intermediate state at pH 5.1. Here, an integrative study is presented investigating how a changing external pH level affects the clostridial acetone-butanol-ethanol (ABE) fermentation pathway. This is of particular interest as the biotechnological production of n-butanol as biofuel has recently returned into the focus of industrial applications. One prerequisite is the furthering of the knowledge of the factors determining the solvent production and their integrative regulations. We have mathematically analysed the influence of pH-dependent specific enzyme activities of branch points of the metabolism on the product formation. This kinetic regulation was compared with transcriptomic regulation regarding gene transcription and the proteomic profile. Furthermore, both regulatory mechanisms were combined yielding a detailed projection of their individual and joint effects on the product formation. The resulting model represents an important platform for future developments of industrial butanol production based on C. acetobutylicum.
An esterase which is encoded within a Thermotoga maritima chromosomal gene cluster for xylan degradation and utilization was characterized after heterologous expression of the corresponding gene in Escherichia coli and purification of the enzyme. The enzyme, designated AxeA, shares amino acid sequence similarity and its broad substrate specificity with the acetyl xylan esterase from Bacillus pumilus, the cephalosporin C deacetylase from Bacillus subtilis, and other (putative) esterases, allowing its classification as a member of carbohydrate esterase family 7. The recombinant enzyme displayed activity with p-nitrophenyl-acetate as well as with various acetylated sugar substrates such as glucose penta-acetate, acetylated oat spelts xylan and DMSO (dimethyl sulfoxide)-extracted beechwood xylan, and with cephalosporin C. Thermotoga maritima AxeA represents the most thermostable acetyl xylan esterase known to date. In a 10 min assay at its optimum pH of 6.5 the enzyme's activity peaked at 90 °C. The inactivation half-life of AxeA at a protein concentration of 0.3 µg µl(-1) in the absence of substrate was about 13 h at 98 °C and about 67 h at 90°C. Differential scanning calorimetry analysis of the thermal stability of AxeA corroborated its extreme heat resistance. A multi-phasic unfolding behaviour was found, with two apparent exothermic peaks at approximately 100-104 °C and 107.5 °C. In accordance with the crystal structure, gel filtration analysis at ambient temperature revealed that the enzyme has as a homohexameric oligomerization state, but a dimeric form was also found.
Mithramycin and chromomycin A(3) are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl-CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.
Two continuous-flow bench-scale bioreactor systems populated by mixed communities of acidophilic sulfate-reducing bacteria were constructed and tested for their abilities to promote the selective precipitation of transition metals (as sulfides) present in synthetic mine waters, using glycerol as electron donor. The objective with the first system (selective precipitation of copper from acidic mine water containing a variety of soluble metals) was achieved by maintaining a bioreactor pH of ≈ 2.2-2.5. The second system was fed with acidic (pH 2.5) synthetic mine water containing 3 mM of both zinc and ferrous iron, and varying concentrations (0.5-30 mM) of aluminium. Selective precipitation of zinc sulfide was possible by operating the bioreactor at pH 4.0 and supplementing the synthetic mine water with 4 mM glycerol. Analysis of the microbial populations in the bioreactors showed that they changed with varying operational parameters, and novel acidophilic bacteria (including one sulfidogen) were isolated from the bioreactors. The acidophilic sulfidogenic bioreactors provided 'proof of principle' that segregation of metals present in mine waters is possible using simple online systems within which controlled pH conditions are maintained. The modular units are versatile and robust, and involve minimum engineering complexity.
Rhodopseudomonas acidophila KU001 was isolated from leather industry effluents and the effect of different cultural conditions on hydrogen production was studied. Anaerobic light induced more hydrogen production than anaerobic dark conditions. Growing cells produced more amounts of hydrogen between 96 and 144 h of incubation. Resting and growing cells preferred a pH of 6.0 ± 0.24 for hydrogen production. Succinate was the most preferred carbon source for the production of hydrogen while citrate was a poor source of carbon. Acetate and malate were also good carbon sources for hydrogen production under anaerobic light. Among the nitrogen sources, R. acidophila preferred ammonium chloride followed by urea for production of hydrogen(.) L-tyrosine was the least preferred nitrogen source by both growing and resting cells.
The bioleaching of metal sulfide has developed into a very important industrial process and understanding the microbial dynamic is key to advancing commercial bioleaching operations. Here we report the first quantitative description of the dynamic of active communities in an industrial bioleaching heap. Acidithiobacillus ferrooxidans was the most abundant during the first part of the leaching cycle, while the abundance of Leptospirillum ferriphilum and Ferroplasma acidiphilum increased with age of the heap. Acidithiobacillus thiooxidans kept constant throughout the leaching cycle, and Firmicutes group showed a low and a patchy distribution in the heap. The Acidiphilium-like bacteria reached their highest abundance corresponding to the amount of autotrophs. The active microorganisms in the leaching system were determined using two RNA-based sensitive techniques. In most cases, the 16S rRNA copy numbers of At. ferrooxidans, L. ferriphilum, At. thiooxidans and F. acidiphilum, was concomitant with the DNA copy numbers, whereas Acidiphilium-like bacteria and some Firmicutes members did not show a clear correlation between 16S rRNA accumulation and DNA copy numbers. However, the prokaryotic acidophile microarray (PAM) analysis showed active members of Alphaproteobacteria in all samples and of Sulfobacillus genus in older ones. Also, new active groups such as Actinobacteria and Acidobacterium genus were detected by PAM. The results suggest that changes during the leaching cycle in chemical and physical conditions, such as pH and Fe(3+)/Fe(2+) ion rate, are primary factors shaping the microbial dynamic in the heap.
Bacterial membranes constitute the first physical barrier against different environmental stresses. Pseudomonas putida DOT-T1E accumulates cyclopropane fatty acids (CFAs) in the stationary phase of growth. In this strain the cfaB gene encodes the main cyclopropane synthase responsible of the synthesis of CFAs, and its expression is mediated by RNA polymerase with sigma factor σ(38). We generated a cfaB mutant of P. putida DOT-T1E and studied its response to solvents, acid pH and other stress conditions such as temperature changes, high osmolarity and the presence of antibiotics or heavy metals in the culture medium. A CfaB knockout mutant was more sensitive to solvent stress than the wild-type strain, but in contrast to Escherichia coli and Salmonella enterica, the P. putida cfaB mutant was as tolerant to acid shock as the wild-type strain. The cfaB mutant was also as tolerant as the parental strain to a number of drugs, antibiotics and other damaging agents.
Enantiomerically pure β-arylalkyl carboxylic acids are important synthetic intermediates for the preparation of a wide range of compounds with biological and pharmacological activities. A library of 83 enzymes isolated from the metagenome was searched for activity in the hydrolysis of ethyl esters of three racemic phenylalkyl carboxylic acids by a microtiter plate-based screening using a pH-indicator assay. Out of these, 20 enzymes were found to be active and were subjected to analytical scale biocatalysis in order to determine their enantioselectivity. The most enantioselective and also enantiocomplementary biocatalysts were then used for preparative scale reactions. Thus, both enantiomers of each of the three phenylalkyl carboxylic acids studied could be obtained in excellent optical purity and high yields.
The position of high-rate anaerobic technology (HR-AnWT) in the wastewater treatment and bioenergy market can be enhanced if the range of suitable substrates is expanded. Analyzing existing technologies, applications and problems, it is clear that, until now, wastewaters with high lipids content are not effectively treated by HR-AnWT. Nevertheless, waste lipids are ideal potential substrates for biogas production, since theoretically more methane can be produced, when compared with proteins or carbohydrates. In this minireview, the classical problems of lipids methanization in anaerobic processes are discussed and new concepts to enhance lipids degradation are presented. Reactors operation, feeding strategies and prospects of technological developments for wastewater treatment are discussed. Long-chain fatty acids (LCFA) degradation is accomplished by syntrophic communities of anaerobic bacteria and methanogenic archaea. For optimal performance these syntrophic communities need to be clustered in compact aggregates, which is often difficult to achieve with wastewaters that contain fats and lipids. Driving the methane production from lipids/LCFA at industrial scale without risk of overloading and inhibition is still a challenge that has the potential for filling a gap in the existing processes and technologies for biological methane production associated to waste and wastewater treatment.
Schematic presentation of the central carbon metabolism of C. glutamicum including pathways for the degradation of carbon sources (glucose, glycerol, D-cellobiose, L-arabinose, D-xylose, mannose, formate, acetate) used for the production of pyruvate, D,L-lactate, 2-ketoisovalerate, 2-ketoglutarate and succinate. Ellipses represent enzymes and transport systems present in C. glutamicum. Rectangles represent heterologous enzymes. Abbreviations: Coding genes are given in brackets. 6PG, 6P-gluconate; 6PGDH (gnd), 6PG dehydrogenase; AHAIR (ilvC), acetohydroxyacid isomeroreductase; AHAS (ilvBN), acetohydroxyacid synthase; AK (ack), acetate kinase; AlaT (alaT), alanine aminotransferase; AraA (araA from E. coli), arabinose isomerase; AraB (araB from E. coli), ribulokinase; AraD (araD from E. coli), L-ribulose-5- phosphate 4-epimerase; AraE (araE from E. coli), L-arabinose transporter; AvtA (avtA), valine-pyruvate aminotransferase; BglA (bglA1, bglA2), phospho-b-glucosidases; BglF (bglF V317A ), mutated PTS permease enabling D-cellobiose import; CtfA (cat), CoA transferase A; DHAD (ilvD), dihydroxyacid dehydratase; DHAP, dihydroxyacetone-P; F1,6P, fructose-1,6P; F6P, fructose-6P; FDH (fdh from Mycobacterium vaccae), formate dehydrogenase; Fum (fum), fumarase; GAP, glyceraldehyde-3P; GAPDH (gapA), GAP dehydrogenase; GlpD (glpD from E. coli), glycerol-3P dehydrogenase; GlpF (glpF from E. coli), glycerol facilitator; GlpK (glpK from E. coli), glycerol kinase; G6P, glucose-6P; G6PDH (zwf, opcA), G6P dehydrogenase; ICD (icd), isocitrate dehydrogenase; ICL (aceA), isocitrate lyase; LDH (native ldhA or ldhA from L. delbrueckii ), L-and D-lactate dehydrogenase respectively; MalE (malE), malic enzyme; MctC (mctC) monocarboxylic acid transporter; Mdh (mdh), malate dehydrogenase; MQO (mqo), malate:quinone oxidoreductase; MS (aceB), malate synthase; ODHC (odhA, aceF, lpd), 2-oxoglutarate dehydrogenase complex; ODx (odx), oxaloacetate decarboxylase; PCx (pyc), pyruvate carboxylase; P, phosphate; PDHC (aceE, aceF, lpd), pyruvate dehydrogenase complex; PEP phosphoenolpyruvate; PEPCk (pck), PEP carboxykinase; PEPCx (ppc), PEP carboxylase; Pyk (pyk), pyruvate kinase; PMI (manA), phosphomannose isomerase; PQO (pqo), pyruvate: quinone oxidoreductase; PTA (pta), phosphotransacetylase; PTS (ptsG, hpr; ptsI), phosphotransferase system; Rpe (rpe), ribulose-5-phosphate epimerase; SDH (sdhABC), succinate dehydrogenase; TA (ilvE), transaminase B; XylA (xylA from E. coli), xylose isomerase; XylB (xylB from E. coli), xylulokinase.  
Schematic presentation of the central carbon metabolism of C. glutamicum ELB-P with the corresponding enzymes and modifications, leading to pyruvate production under aerobic conditions and reductive succinate production under anaerobic conditions. For most abbreviations see legend to Fig. 1. Further abbreviations: BCAAs, branched-chain amino acids; LdhA, NAD + -dependent L-lactate dehydrogenase; SucE, succinate exporter; TCA, tricarboxylic acid. Down arrow at AHAS indicates decreased activity of the truncated AHAS derivative, crosses indicate inactivation of the enzyme by deletion of the respective gene. Dotted arrows indicate pathways not present due to gene inactivation, grey arrows indicate pathways not used in C. glutamicum ELB-P.  
Schematic presentation of the central carbon metabolism of C. glutamicum BOL-3/ pAN6-gap during anaerobic succinate production . For most abbreviations see legend to Fig. 1. Further abbreviations: BCAAs, branched-chain amino acids; LdhA, NAD + dependent L-lactate dehydrogenase; SucE, succinate exporter; TCA, tricarboxylic acid. Dark ellipses indicate homologous/ heterologous enzymes, crosses indicate inactivation of the enzyme by deletion of the respective gene. Dotted arrows indicate pathways not present due to gene inactivation, grey arrows indicate pathways not used in C. glutamicum BOL-3/pAN6-gap.  
Growth, glucose consumption, and product formation (A) and acetate consumption and the course of the pO2 (B) during a representative pH-controlled tri-phasic fed-batch cultivation of C. glutamicum ELB-P in a 400 ml bioreactor with minimal medium, initially containing 4% (w/v) glucose, 1% (w/v) acetate and 6 mM L-alanine. (A) , growth; , glucose; , pyruvate; , succinate; , malate; , lactate. (B) Black line, pO2; grey line, acetate. Roman numerals indicate (I) aerobic growth phase, (II) self-induced microaerobic phase, and (III) oxygen deprivation by CO2 gassing. The pO2 peak at the end of the microaerobic phase (marked with the asterisk in 4B) indicated the end of aerobic growth, as O2 consumption stopped, leading to increasing DO in the medium. Immediately, aeration was replaced by CO2 sparging and the production phase started. A batch of glucose at the beginning of phase III should ensure carbon availability for succinate production. At least five independent fermentations were performed, showing comparable results.  
The shortage of oil resources, the steadily rising oil prices and the impact of its use on the environment evokes an increasing political, industrial and technical interest for development of safe and efficient processes for the production of chemicals from renewable biomass. Thus, microbial fermentation of renewable feedstocks found its way in white biotechnology, complementing more and more traditional crude oil-based chemical processes. Rational strain design of appropriate microorganisms has become possible due to steadily increasing knowledge on metabolism and pathway regulation of industrially relevant organisms and, aside from process engineering and optimization, has an outstanding impact on improving the performance of such hosts. Corynebacterium glutamicum is well known as workhorse for the industrial production of numerous amino acids. However, recent studies also explored the usefulness of this organism for the production of several organic acids and great efforts have been made for improvement of the performance. This review summarizes the current knowledge and recent achievements on metabolic engineering approaches to tailor C. glutamicum for the bio-based production of organic acids. We focus here on the fermentative production of pyruvate, l-and d-lactate, 2-ketoisovalerate, 2-ketoglutarate, and succinate. These organic acids represent a class of compounds with manifold application ranges, e.g. in pharmaceutical and cosmetics industry, as food additives, and economically very interesting, as precursors for a variety of bulk chemicals and commercially important polymers. Funding Information Work in the laboratories of the authors was supported by the Fachagentur Nachwachsende Rohstoffe (FNR) of the Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV; FNR Grants 220-095-08A and 220-095-08D; Bio-ProChemBB project, ERA-IB programme), by the Deutsche Bundesstiftung Umwelt (DBU Grant AZ13040/05) and the Evonik Degussa AG.
Bacillus subtilis is a widespread and diverse bacterium t exhibits a remarkable intraspecific diversity of the ComQXPA quorum-sensing (QS) system. This manifests in the existence of distinct communication groups (pherotypes) that can efficiently communicate within a group, but not between groups. Similar QS diversity was also found in other bacterial species, and its ecological and evolutionary meaning is still being explored. Here we further address the ComQXPA QS diversity among isolates from the tomato rhizoplane, a natural habitat of B. subtilis, where these bacteria likely exist in their vegetative form. Because this QS system regulates production of anti-pathogenic and biofilm-inducing substances such as surfactins, knowledge on cell-cell communication of this bacterium within rhizoplane is also important from the biocontrol perspective. We confirm the presence of pherotype diversity within B. subtilis strains isolated from a rhizoplane of a single plant. We also show that B. subtilis rhizoplane isolates show a remarkable diversity of surfactin production and potential plant growth promoting traits. Finally, we discover that effects of surfactin deletion on biofilm formation can be strain specific and unexpected in the light of current knowledge on its role it this process. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
This work demonstrates that Acinetobacter radioresistens strain S13 during the growth on medium supplemented with long-chain alkanes as the sole energy source expresses almA gene coding for a Baeyer-Villiger monooxygenase (BVMO) involved in alkanes subterminal oxidation. Phylogenetic analysis placed the sequence of this novel BVMO in the same clade of the prodrug activator ethionamide monooxygenase (EtaA) and it bears only a distant relation to the other known class I BVMO proteins. In silico analysis of the 3D model of the S13 BVMO generated by homology modelling also supports the similarities with EtaA by binding ethionamide to the active site. In vitro experiments carried out with the purified enzyme confirm that this novel BVMO is indeed capable of typical Baeyer-Villiger reactions as well as oxidation of the prodrug ethionamide.
The members of the Lactobacillus genus are widely used in the food and feed industry and show a remarkable ecological adaptability. Several Lactobacillus strains have been marketed as probiotics as they possess health-promoting properties for the host. In the present study, we used two complementary next-generation sequencing technologies to deduce the genome sequences of two Lactobacillus casei strains LcA and LcY, which were isolated from the products Actimel and Yakult, commercialized as probiotics. The LcA and LcY draft genomes have, respectively, an estimated size of 3067 and 3082Mb and a G+C content of 46.3%. Both strains are close to identical to each other and differ by no more than minor chromosomal re-arrangements, substitutions, insertions and deletions, as evident from the verified presence of one insertion-deletion (InDel) and only 29 single-nucleotide polymorphisms (SNPs). In terms of coding capacity, LcA and LcY are predicted to encode a comparable exoproteome, indicating that LcA and LcY are likely to establish similar interactions with human intestinal cells. Moreover, both L.caseiLcA and LcY harboured a 59.6kb plasmid that shared high similarities with plasmids found in other L.casei strains, such as W56 and BD-II. Further analysis revealed that the L.casei plasmids constitute a good evolution marker within the L.casei species. The plasmids of the LcA and LcY strains are almost identical, as testified by the presence of only three verified SNPs, and share a 3.5kb region encoding a remnant of a lactose PTS system that is absent from the plasmids of W56 and BD-II but conserved in another smaller L.casei plasmid (pLC2W). Our observations imply that the results obtained in animal and human experiments performed with the Actimel and Yakult strains can be compared with each other as these strains share a very recent common ancestor. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
Natural products are traditionally the main source of drug leads. In particular, many antitumour compounds are either natural products or derived from them. However, the search for novel antitumour drugs active against untreatable tumours, with fewer side-effects or with enhanced therapeutic efficiency, is a priority goal in cancer chemotherapy. Microorganisms, particularly actinomycetes, are prolific producers of bioactive compounds, including antitumour drugs, produced as secondary metabolites. Structural genes involved in the biosynthesis of such compounds are normally clustered together with resistance and regulatory genes, which facilitates the isolation of the gene cluster. The characterization of these clusters has represented, during the last 25 years, a great source of genes for the generation of novel derivatives by using combinatorial biosynthesis approaches: gene inactivation, gene expression, heterologous expression of the clusters or mutasynthesis. In addition, these techniques have been also applied to improve the production yields of natural and novel antitumour compounds. In this review we focus on some representative antitumour compounds produced by actinomycetes covering the genetic approaches used to isolate and validate their biosynthesis gene clusters, which finally led to generating novel derivatives and to improving the production yields.
Top-cited authors
Willy Verstraete
  • Ghent University
Juan L Ramos
  • Spanish National Research Council
Engy Ahmed
  • Stockholm University
Sara Holmstrom
  • Norconsult AB
Ren Wei
  • University of Greifswald