Consortia modulation of the stress response: proteomic analysis of single strain versus mixed culture

The high complexity of naturally occurring microbial communities is the major drawback limiting the study of these important biological systems. In this study, a comparison between pure cultures of Pseudomonas reinekei sp. strain MT1 and stable community cultures composed of MT1 plus the addition of Achromobacter xylosoxidans strain MT3 (in a steady‐state proportion 9:1) was used as a model system to study bacterial interactions that take place under simultaneous chemical and oxidative stress. Both are members of a real community isolated from a polluted sediment by enrichment in 4‐chlorosalicylate (4CS). The analysis of dynamic states was carried out at the proteome, metabolic profile and population dynamic level. Differential protein expression was evaluated under exposure to 4CS and high concentrations of toxic intermediates (4‐chlorocatechol and protoanemonin), including proteins from several functional groups and particularly enzymes of aromatic degradation pathways and outer membrane proteins. Remarkably, 4CS addition generated a strong oxidative stress response in pure strain MT1 culture led by alkyl hydroperoxide reductase, while the community showed an enhanced central metabolism response, where A. xylosoxidans MT3 helped to prevent toxic intermediate accumulation. A significant change in the outer membrane composition of P. reinekei MT1 was observed during the chemical stress caused by 4CS and in the presence of A. xylosoxidans MT3, highlighting the expression of the major outer membrane protein OprF, tightly correlated to 4CC concentration profile and its potential detoxification role.     

High tolerance to glycerol and high production of 1,3‐propanediol in batch fermentations by microbial consortium from marine sludge

1,3‐Propanediol (1,3‐PD) is a versatile bulk chemical and widely used as a monomer to synthesis polymers, such as polyesters, polyethers and polyurethanes. 1,3‐PD can be produced by microbial fermentation with the advantages of the environmental protection and sustainable development. Low substrate tolerance and wide by‐product profile limit microbial production of 1,3‐PD by Klebsiella pneumonia on industrial scale. In this study, microbial consortia were investigated to overcome some disadvantages of pure fermentation by single strain. Microbial consortium named DL38 from marine sludge gave the best performance. Its bacterial community composition was analyzed by 16S rRNA gene amplicon high‐throughput sequencing and showed that Enterobacteriaceae was the most abundant family. Compared with three K. pneumonia strains isolated from DL38, the microbial consortium could grow well at an initial glycerol concentration of 200 g/L to produce 81.40 g/L of 1,3‐PD with a yield of 0.63 mol/mol. This initial glycerol concentration is twice the highest concentration by single isolated strain and more than the critical value (188 g/L) extrapolated from the fermentation kinetics for K. pneumonia. On the other hand, a small amount of by‐products were produced in batch fermentation of microbial consortium DL38,  especially no 2,3‐butanediol detected. The mixed culture of strain W3, Y5 and Y1 improved the tolerance to glycerol and changed the metabolite profile of single strain W3. The batch fermentation with the natural proportion (W3: Y5: Y1 = 208: 82: 17) was superior to that with other proportions and single strain. This study showed that microbial consortium DL38 possessed excellent substrate tolerance, narrow by‐product profile and attractive potential for industrial production of 1,3‐PD.     

Bacterial consortium proteomics under 4‐chlorosalicylate carbon‐limiting conditions

In this study, the stable consortium composed by Pseudomonas reinekei strain MT1 and Achromobacter xylosoxidans strain MT3 (cell numbers in proportion 9:1) was under investigation to reveal bacterial interactions that take place under severe nutrient‐limiting conditions. The analysis of steady states in continuous cultures was carried out at the proteome, metabolic profile, and population dynamic levels. Carbon‐limiting studies showed a higher metabolic versatility in the community through upregulation of parallel catabolic enzymes (salicylate 5‐hydroxylase and 17‐fold on 2‐keto‐4‐pentenoate hydratase) indicating a possible alternative carbon routing in the upper degradation pathway highlighting the effect of minor proportions of strain MT3 over the major consortia component strain MT1 with a significant change in the expression levels of the enzymes of the mainly induced biodegradation pathway such as salicylate 1‐hydroxylase and catechol 1,2‐dioxygenase together with important changes in the outer membrane composition of P. reinekei MT1 under different culture conditions. The study has demonstrated the importance of the outer membrane as a sensing/response protective barrier caused by interspecies interactions highlighting the role of the major outer membrane proteins OprF and porin D in P. reinekei sp. MT1 under the culture conditions tested.     

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Consolidated bioprocessing (CBP) by using microbial consortium was considered as a promising approach to achieve direct biofuel production from lignocellulose. In this study, the interaction mechanism of microbial consortium consisting of Thermoanaerobacterium thermosaccharolyticum M5 and Clostridium acetobutylicum NJ4 was analyzed, which could achieve efficient butanol production from xylan through CBP. Strain M5 possesses efficient xylan degradation capability, as 19.73 g/L of xylose was accumulated within 50 hr. The efficient xylose utilization capability of partner strain NJ4 could relieve the substrate inhibition to hydrolytic enzymes of xylanase and xylosidase secreted by strain M5. In addition, the earlier solventogenesis of strain NJ4 was observed due to the existence of butyrate generated by strain M5. The mutual interaction of these two strains finally gave 13.28 g/L of butanol from 70 g/L of xylan after process optimization, representing a relatively high butanol production from hemicellulose. Moreover, 7.61 g/L of butanol was generated from untreated corncob via CBP. This successfully constructed microbial consortium exhibits efficient cooperation performance on butanol production from lignocellulose, which could provide a platform for the emerging butanol production from lignocellulose.     

Isolation of bacterial consortia for degradation of p-nitrophenol from agricultural soil

bacterial consortium has been isolated containing Pseudomonas spp. strains S1 and S2, which was able to degrade p‐nitrophenol (PNP). The strains were isolated from agricultural soil contaminated with organophosphorus pesticides. Pseudomonas spp. strain S2 could convert p‐nitrophenol to 4‐nitrocatechol (4NC) after pre‐exposure to phenol, when PNP was used as the only carbon source in the medium. Pseudomonas spp. strain S2, when mixed with strain S1 in the ratio 1:5 respectively, decolorised PNP completely.     

Performance and microbial diversity of palm oil mill effluent microbial fuel cell

Aim: To evaluate the bioenergy generation and the microbial community structure from palm oil mill effluent using microbial fuel cell. Methods and Results: Microbial fuel cells enriched with palm oil mill effluent (POME) were employed to harvest bioenergy from both artificial wastewater containing acetate and complex POME. The microbial fuel cell (MFC) showed maximum power density of 3004 mW m^?2 after continuous feeding with artificial wastewater containing acetate substrate. Subsequent replacement of the acetate substrate with complex substrate of POME recorded maximum power density of 622 mW m^?2. Based on 16S rDNA analyses, relatively higher abundance of Deltaproteobacteria (88·5%) was detected in the MFCs fed with acetate artificial wastewater as compared to POME. Meanwhile, members of Gammaproteobacteria, Epsilonproteobacteria and Betaproteobacteria codominated the microbial consortium of the MFC fed with POME with 21, 20 and 18·5% abundances, respectively. Conclusions: Enriched electrochemically active bacteria originated from POME demonstrated potential to generate bioenergy from both acetate and complex POME substrates. Further improvements including the development of MFC systems that are able to utilize both fermentative and nonfermentative substrates in POME are needed to maximize the bioenergy generation. Significance and Impact of the Study: A better understanding of microbial structure is critical for bioenergy generation from POME using MFC. Data obtained in this study improve our understanding of microbial community structure in conversion of POME to electricity.     

Two novel decarboxylase genes play a key role in the stereospecific catabolism of dehydrodiconiferyl alcohol in Sphingobium sp. strain SYK‐6

Sphingobium sp. strain SYK‐6 is able to use a phenylcoumaran‐type biaryl, dehydrodiconiferyl alcohol (DCA), as a sole source of carbon and energy. In SYK‐6 cells, the alcohol group of the B‐ring side chain of DCA was first oxidized to the carboxyl group, and then the alcohol group of the A‐ring side chain was oxidized to generate 5‐(2‐carboxyvinyl)‐2‐(4‐hydroxy‐3‐methoxyphenyl)‐7‐methoxy‐2,3‐dihydrobenzofuran‐3‐carboxylate (DCA‐CC). We identified phcF, phcG and phcH, which conferred the ability to convert DCA‐CC into 3‐(4‐hydroxy‐3‐(4‐hydroxy‐3‐methoxystyryl)‐5‐methoxyphenyl)acrylate (DCA‐S) in a host strain. These genes exhibited no significant sequence similarity with known enzyme genes, whereas phcF and phcG, which contain a DUF3237 domain of unknown function, showed 32% amino acid sequence identity with each other. The DCA‐CC conversion activities were markedly decreased by disruption of phcF and phcG, indicating that phcF and phcG play dominant roles in the conversion of DCA‐CC. Purified PhcF and PhcG catalysed the decarboxylation of the A‐ring side chain of DCA‐CC, producing DCA‐S, and showed enantiospecificity towards (+)‐ and (–)‐DCA‐CC respectively. PhcF and PhcG formed homotrimers, and their K_m for DCA‐CC were determined to be 84 μM and 103 μM, and V_max were 307 μmol?min^?1?mg^?1 and 137 μmol?min^?1?mg^?1 respectively. In conclusion, PhcF and PhcG are enantiospecific decarboxylases involved in phenylcoumaran catabolism.     

Degradation of biphenyl by methanogenic microbial consortium

Biphenyl was readily degraded and mineralized to CO₂ and CH₄ by a PCB-dechlorinating anaerobic microbial consortium. Degradation occurred when biphenyl was supplied as a sole source of carbon or as a co-metabolic substrate together with glucose and methanol. p-Cresol was detected and confirmed by mass spectroscopy as a transient intermediate. Production of ¹⁴ C-CO₂ and ¹⁴C-CH₄ from ¹⁴C-biphenyl was observed in the approximate ratio of 1:2. The results indicated the existence of novel pathways for biphenyl degradation in a natural anaerobic microbial community.     

New approaches in bioprocess‐control: Consortium guidance by synthetic cell‐cell communication based on fungal pheromones

Bioconversions in industrial processes are currently dominated by single‐strain approaches. With the growing complexity of tasks to be carried out, microbial consortia become increasingly advantageous and eventually may outperform single‐strain fermentations. Consortium approaches benefit from the combined metabolic capabilities of highly specialized strains and species, and the inherent division of labor reduces the metabolic burden for each strain while increasing product yields and reaction specificities. However, consortium‐based designs still suffer from a lack of available tools to control the behavior and performance of the individual subpopulations and of the entire consortium. Here, we propose to implement novel control elements for microbial consortia based on artificial cell–cell communication via fungal mating pheromones. Coupling to the desired output is mediated by pheromone‐responsive gene expression, thereby creating pheromone‐dependent communication channels between different subpopulations of the consortia. We highlight the benefits of artificial communication to specifically target individual subpopulations of microbial consortia and to control e.g. their metabolic profile or proliferation rate in a predefined and customized manner. Due to the steadily increasing knowledge of sexual cycles of industrially relevant fungi, a growing number of strains and species can be integrated into pheromone‐controlled sensor‐actor systems, exploiting their unique metabolic properties for microbial consortia approaches.     

Mineralization of low-chlorinated biphenyls by Burkholderia sp. strain LB400 and by a two-membered consortium upon directed interspecies transfer of chlorocatechol pathway genes

The biphenyl-mineralizing bacterium Burkholderia sp. strain LB400 also utilized 3-chloro-, 4-chloro-, 2,3′-dichloro- and 2,4′-dichlorobiphenyl for growth. By the attack of the initial enzyme a chlorine was eliminated dioxygenolytically from position 2 of one of the aromatic rings when hydrogens of both were substituted by chlorine. The strain mineralized 3-chloro- and 2,3′-dichlorobiphenyl via the central intermediate 3-chlorobenzoate through its chlorocatechol pathway enzymes, but excreted stoichiometric amounts of 4-chlorobenzoate from 4-chloro- and 2,4^′-dichlorobiphenyl. These two compounds were mineralized by a co-culture of strain LB400 and a derivative of the (methyl-) benzoate-degrading strain Pseudomonas putida mt-2 (TOL). The complete degradation was achieved upon transfer of a cluster of at least five genes, encoding the regulated chlorocatechol pathway operon, from strain LB400 to strain mt-2. This transfer was demonstrated by the polymerase chain reaction. Received: 15 April 1998 / Received revision: 12 June 1998 / Accepted: 19 June 1998     

Functional consortium for denitrifying sulfide removal process

Denitrifying sulfide removal (DSR) process simultaneously converts sulfide, nitrate, and chemical oxygen demand from industrial wastewaters to elemental sulfur, nitrogen gas, and carbon dioxide, respectively. This investigation utilizes a dilution-to-extinction approach at 10⁻² to 10⁻⁶ dilutions to elucidate the correlation between the composition of the microbial community and the DSR performance. In the original suspension and in 10⁻² dilution, the strains Stenotrophomonas sp., Thauera sp., and Azoarcus sp. are the heterotrophic denitrifiers and the strains Paracoccus sp. and Pseudomonas sp. are the sulfide-oxidizing denitrifers. The 10⁻⁴ dilution is identified as the functional consortium for the present DSR system, which comprises two functional strains, Stenotrophomonas sp. strain Paracoccus sp. At 10⁻⁶ dilution, all DSR performance was lost. The functions of the constituent cells in the DSR granules were discussed based on data obtained using the dilution-to-extinction approach.     

A brief review of microbial arsenate respiration

In this review, we summarize the important recent findings relating to arsenate respi‐ration by bacteria. A brief discussion of freshwater arsenic cycling is provided, with attention placed on the microbial contributions to this cycle. The basic evidence for microbial growth on arsenate is presented for studies with both consortia and isolates, followed by a summary of the physiology and phytogeny of four arsenate‐respiring bac‐teria: Chrysiogenes arsenatis strain BAL‐1^T, Desulfotomaculum auripigmentum strain OREX‐4, Sulfurospirillum arsenophilus strain MIT‐13, and S. barnesii strain SES‐3. Drawing on biochemical studies of the arsenate reductasefrom S. barnesii strain SES‐3, a preliminary model for growth on arsenate is proposed. We conclude with a discussion of the importance of microbial arsenate reduction in the environment.     

A single amino acid substitution in the MurF UDP‐MurNAc‐pentapeptide synthetase renders Streptococcus pneumoniae dependent on CO2 and temperature

The respiratory tract pathogen Streptococcus pneumoniae encounters different levels of environmental CO₂ during transmission, host colonization and disease. About 8% of all pneumococcal isolates are capnophiles that require CO₂‐enriched growth conditions. The underlying molecular mechanism for caphnophilic behaviour, as well as its biological function is unknown. Here, we found that capnophilic S. pneumoniae isolates from clonal complex (CC) 156 (i.e. Spain^9V‐3 ancestry) and CC344 (i.e. Norway^NT‐42 ancestry) have a valine at position 179 in the MurF UDP‐MurNAc‐pentapeptide synthetase. At ≤ 30°C, the growth characteristics of capnophilic and non‐capnophilic CC156 strains were equal, but at > 30°C growth and survival of MurF^V179 strains was dependent on > 0.1% CO₂‐enriched conditions. Expression of MurF^V179 in S. pneumoniae R6 and G54 rendered these, otherwise non‐capnophilic strains, capnophilic. Time‐lapse microscopy revealed that a capnophilic CC156 strain undergoes rapid autolysis upon exposure to CO₂‐poor conditions at 37°C, and staining with fluorescently labelled vancomycin showed a defect in de novo cell wall synthesis. In summary, in capnophilic S. pneumoniae strains from CC156 and CC344 cell wall synthesis is placed under control of environmental CO₂ levels and temperature. This mechanism might represent a novel strategy of the pneumococcus to rapidly adapt and colonize its host under changing environmental conditions.     

Pathways for 3-chloro- and 4-chlorobenzoate degradationin Pseudomonas aeruginosa 3mT

A bacterial isolate, Pseudomonas aeruginosa 3mT, exhibited the ability to degrade high concentrations of 3-chlorobenzoate (3-CBA, 8 g l^-1) and 4-chlorobenzoate (4-CBA 12 g l^-1) (Ajithkumar 1998). In this study, by delineating the initial biochemical steps involved in the degradation of these compounds, we investigated how this strain can do so well. Resting cells, permeabilised cells as well as cell-free extracts failed to dechlorinate both 3-CBA and 4-CBA under anaerobic conditions, whereas the former two readily degraded both compounds under aerobic conditions. Accumulation of any intermediary metabolite was not observed during growth as well as reaction with resting cells under highly aerated conditions. However, on modification of reaction conditions, 3-chlorocatechol (3-CC) and 4-chlorocatechol (4-CC) accumulated in 3-CBA and 4-CBA flasks, respectively. Fairly high titres of pyrocatechase II (chlorocatechol 1,2-dioxygenase) activity were obtained in extracts of cells grown on 3-CBA and 4-CBA. Meta-pyrocatechase (catechol 2,3-dioxygenase) activity against4-CC and catechol, but not against 3-CC, was also detected in low titres. Accumulation of small amounts of 2-chloro-5-hydroxy muconic semialdehyde, the meta-cleavage product of 4-CC, was detected in the medium, when 4-CBA concentration was 4 mM or greater, indicating the presence of a minor meta-pathway in strain 3mT. However, 3-CBA exclusively, and more than 99% of 4-CBA were degraded through the formation of the respective chlorocatechol, via a modified ortho-pathway. This defies the traditional view that the microbes that follow chlorocatechol pathways are not very good degraders of chlorobenzoates. 4-Hydroxybenzoatewas readily (and 3-hydroxybenzoate to a lesser extent) degraded by the strain, through the formation of protocatechuate and gentisate, respectively, as intermediary dihydroxy metabolites.     

Structure of the Microbial Communities in Coniferous Forest Soils in Relation to Site Fertility and Stand Development Stage

Abstract The structure, biomass, and activity of the microbial community in the humus layer of boreal coniferous forest stands of different fertility were studied. The Scots pine dominated CT (Calluna vulgaris type) represented the lowest fertility, while VT (Vaccinium vitis-idaéa type), MT (Vaccinium myrtillus type), and OMT (Oxalis acetocella–Vaccinium myrtillus type) following this order, were more fertile types. The microbial community was studied more closely by sampling a succession gradient (from a treeless area to a 180-years-old Norway spruce stand) at the MT type site. The phospholipid fatty acid (PLFA) analysis revealed a gradual shift in the structure of the microbial community along the fertility gradient even though the total microbial biomass and respiration rate remained unchanged. The relative abundance of fungi decreased and that of bacteria increased with increasing fertility. The structure of the bacterial community also changed along the fertility gradient. Irrespective of a decrease in fungal biomass and change in bacterial community structure after clear-cutting, the PLFA analysis did not show strong differences in the microbial communities in the stands of different age growing on the MT type site. The spatial variation in the structure of the microbial community was studied at a MT type site. Semivariograms indicated that the bacterial biomass, the ratio between the fungal and bacterial biomasses, and the relative amount of PLFA 16:1ω5 were spatially autocorrelated within distances around 3 to 4 m. The total microbial and fungal biomasses were autocorrelated only up to 1 m. The spatial distribution of the humus microbial community was correlated mainly with the location of the trees, and consequently, with the forest floor vegetation. Received: 9 November 1998; Accepted: 26 April 1999     

Constructing E. coli Co‐Cultures for De Novo Biosynthesis of Natural Product Acacetin

Modular co‐culture engineering is an emerging approach for biosynthesis of complex natural products. In this study, microbial co‐cultures composed of two and three Escherichia coli strains, respectively, are constructed for de novo biosynthesis of flavonoid acacetin, a value‐added natural compound possessing numerous demonstrated biological activities, from simple carbon substrate glucose. To this end, the heterologous biosynthetic pathway is divided into different modules, each of which is accommodated in a dedicated E. coli strain for functional expression. After the optimization of the inoculation ratio between the constituent strains, the engineered co‐cultures show a 4.83‐fold improvement in production comparing to the mono‐culture controls. Importantly, cultivation of the three‐strain co‐culture in shake flasks result in the production of 20.3 mg L^?1 acacetin after 48 h. To the authors' knowledge, this is the first report on acacetin de novo biosynthesis in a heterologous microbial host. The results of this work confirm the effectiveness of modular co‐culture engineering for complex flavonoid biosynthesis.     

ALLOPOLYPLOIDY IN NATURAL AND CULTIVATED POPULATIONS OF PORPHYRA (BANGIALES,RHODOPHYTA)1

To confirm whether allopolyploidy occurs in samples of previously identified Porphyra yezoensis Ueda, P. tenera Kjellm., and P. yezoensis × P. tenera from natural and cultivated populations, we examined these samples by using PCR‐RFLP and microsatellite analyses of multiple nuclear and chloroplast regions [nuclear regions: type II DNA topoisomerase gene (TOP2), actin‐related protein 4 gene (ARP4), internal transcribed spacer (ITS) rDNA and three microsatellite loci; chloroplast region: RUBISCO spacer]. Except for the ITS region, these multiple nuclear markers indicated that the wild strain MT‐1 and the cultivated strain 90‐02 (previously identified as P. yezoensis × P. tenera and cultivated P. tenera, respectively) are heterozygous and possess both genotypes of P. tenera and P. yezoensis in the conchocelis phase. Furthermore, gametophytic blades of two pure lines, HG‐TY1 and HG‐TY2 (F₁ strains of MT‐1 and 90‐02, respectively), were also heterozygous, and six chromosomes per single cell could be observed in each blade of the two pure lines. These results demonstrate that allopolyploidy occurs in Porphyra strains derived from both natural and cultivated populations, even though ITS genotypes of these strains showed homogenization toward one parental ITS.     

Genetic and hormonal control of melanization in reddish–brown and albino mutants in the desert locust Schistocerca gregaria

The genetic and hormonal control of body colouration is investigated using two recessive genetic mutant strains, the reddish–brown (RB) mutant and an albino mutant, as well as a normal (pigmented) strain of the desert locust Schistocerca gregaria. The colour patterns of the RB nymphs are similar to those of a normal strain, although the intensity of the melanization is weaker in the former. Reciprocal crosses between the RB and albino mutants produce only normal phenotypes in the F₁ generation. In the F₂ generation, the normal, RB and albino phenotypes appear in a ratio of 9 : 3 : 4, indicating that two Mendelian units might determine the appearance of dark body colour and the intensity of melanization, respectively. In other words, at least two steps of regulation might be involved in the expression of body colour. Injections of [His⁷]‐corazonin, a neuropeptide inducing dark colour in this locust, fail to induce dark colour in albino nymphs but show a dose‐dependent darkening in RB nymphs in the range, 10 pmol to 1 nmol. Some RB nymphs become indistinguishable from normal individuals after injection of the peptide. Implantation of corpora cardiaca (CC) taken from RB mutants into other RB individuals induces darkening in the latter and CC from RB, albino and normal strains have similar dark colour‐inducing activity when implanted into albino Locusta migratoria. These results suggest the possibility that the RB mutant gene regulates the intensity of melanization, possibly through controlling the pathway of pigment biosynthesis associated with [His⁷]‐corazonin.     

High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use

Most microorganisms remain uncultivated, and typically their ecological roles must be inferred from diversity and genomic studies. To directly measure functional roles of uncultivated microbes, we developed Chip-stable isotope probing (SIP), a high-sensitivity, high-throughput SIP method performed on a phylogenetic microarray (chip). This approach consists of microbial community incubations with isotopically labeled substrates, hybridization of the extracted community rRNA to a microarray and measurement of isotope incorporation—and therefore substrate use—by secondary ion mass spectrometer imaging (NanoSIMS). Laboratory experiments demonstrated that Chip-SIP can detect isotopic enrichment of 0.5 atom % ¹³C and 0.1 atom % ¹⁵N, thus permitting experiments with short incubation times and low substrate concentrations. We applied Chip-SIP analysis to a natural estuarine community and quantified amino acid, nucleic acid or fatty acid incorporation by 81 distinct microbial taxa, thus demonstrating that resource partitioning occurs with relatively simple organic substrates. The Chip-SIP approach expands the repertoire of stable isotope-enabled methods available to microbial ecologists and provides a means to test genomics-generated hypotheses about biogeochemical function in any natural environment.