Towards the rate limit of heterologous biotechnological reactions in recombinant cyanobacteria.

BACKGROUND: Cyanobacteria have emerged as highly efficient organisms for the production of chemicals and biofuels. Yet, the productivity of the cell has been low for commercial application. Cyanobacterial photobiotransformations utilize photosynthetic electrons to form reducing equivalents, such as NADPH-to-fuel biocatalytic reactions. These photobiotransformations are a measure to which extent photosynthetic electrons can be deviated toward heterologous biotechnological processes, such as the production of biofuels. By expressing oxidoreductases, such as YqjM from Bacillus subtilis in Synechocystis sp. PCC 6803, a high specific activity was obtained in the reduction of maleimides. Here, we investigated the possibility to accelerate the NAD(P)H-consuming redox reactions by addition of carbohydrates as exogenous carbon sources such as D-Glucose under light and darkness. RESULTS: A 1.7-fold increase of activity (150 µmol min-1 gDCW-1) was observed upon addition of D-Glucose at an OD750 = 2.5 (DCW = 0.6 g L-1) in the biotransformation of 2-methylmaleimide. The stimulating effect of D-Glucose was also observed at higher cell densities in light and dark conditions as well as in the reduction of other substrates. No increase in both effective photosynthetic yields of Photosystem II and Photosystem I was found upon D-Glucose addition. However, we observed higher NAD(P)H fluorescence when D-Glucose was supplemented, suggesting increased glycolytic activity. Moreover, the system was scaled-up (working volume of 200 mL) in an internally illuminated Bubble Column Reactor exhibiting a 2.4-fold increase of specific activity under light-limited conditions. CONCLUSIONS: Results show that under photoautotrophic conditions at a specific activity of 90 µmol min-1 gDCW-1, the ene-reductase YqjM in Synechocystis sp. PCC 6803 is not NAD(P)H saturated, which is an indicator that an increase of the rates of heterologous electron consuming processes for catalysis and biofuel production will require funnelling further reducing power from the photosynthetic chain toward heterologous processes.

Extracellular vesicles as an alternative copper-secretion mechanism in bacteria.

Metal homeostasis is fundamental for optimal performance of cell metabolic pathways. Over the course of evolution, several systems emerged to warrant an intracellular metal equilibrium. When exposed to growth-challenging copper concentrations, Gram-negative bacteria quickly activate copper-detoxification mechanisms, dependent on transmembrane-protein complexes and metallochaperones that mediate metal efflux. Here, we show that vesiculation is also a common bacterial response mechanism to high copper concentrations, and that extracellular vesicles (EVs) play a role in transporting copper. We present evidence that bacteria from different ecological niches release copious amounts of EVs when exposed to copper. Along with the activation of the classical detoxification systems, we demonstrate that copper-stressed cells of the cyanobacterium Synechocystis sp. PCC6803 release EVs loaded with the copper-binding metallochaperone CopM. Under standard growth conditions, CopM-loaded EVs could also be isolated from a Synechocystis strain lacking a functional TolC-protein, which we characterize here as exhibiting a copper-sensitive phenotype. Analyses of Synechocystis tolC-mutant's EVs isolated from cells cultivated under standard conditions indicated the presence of copper therein, in significantly higher levels as compared to those from the wild-type. Altogether, these results suggest that release of EVs in bacteria represent a novel copper-secretion mechanism, shedding light into alternative mechanisms of bacterial metal resistance.

CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803.

The use of the versatile cyanobacterial extracellular polymeric substances (EPS) for biotechnological/biomedical applications implies an extensive knowledge of their biosynthetic pathways to improve/control polymer production yields and characteristics. The multiple copies of EPS-related genes, scattered throughout cyanobacterial genomes, adds another layer of complexity, making these studies challenging and time-consuming. Usually, this issue would be tackled by generating deletion mutants, a process that in cyanobacteria is also hindered by the polyploidy. Thus, the use of the CRISPRi multiplex system constitutes an efficient approach to addressing this redundancy. Here, three putative Synechocystis sp. PCC 6803 kpsM homologues (slr0977, slr2107, and sll0574) were repressed using this methodology. The characterization of the 3-sgRNA mutant in terms of fitness/growth and total carbohydrates, released and capsular polysaccharides, and its comparison with previously generated single knockout mutants pointed towards Slr0977 being the key KpsM player in Synechocystis EPS production. This work validates CRISPRi as a powerful tool to unravel cyanobacterial complex EPS biosynthetic pathways expediting this type of studies.

Design and Validation of Tools for Microbial Synthetic Biology Applications.

Synthetic Biology (SynBio) is a multidisciplinary field that brings together science, technology and engineering to expedite the design, creation and modification of genetic materials to be applied in living organisms or in vitro systems [...].

Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number.

BACKGROUND: Synechocystis sp. PCC 6803 provides a well-established reference point to cyanobacterial metabolic engineering as part of basic photosynthesis research, as well as in the development of next-generation biotechnological production systems. This study focused on expanding the current knowledge on genomic integration of expression constructs in Synechocystis, targeting a range of novel sites in the chromosome and in the native plasmids, together with established loci used in literature. The key objective was to obtain quantitative information on site-specific expression in reference to replicon copy numbers, which has been speculated but never compared side by side in this host. RESULTS: An optimized sYFP2 expression cassette was successfully integrated in two novel sites in Synechocystis chromosome (slr0944; sll0058) and in all four endogenous megaplasmids (pSYSM/slr5037-slr5038; pSYSX/slr6037; pSYSA/slr7023; pSYSG/slr8030) that have not been previously evaluated for the purpose. Fluorescent analysis of the segregated strains revealed that the expression levels between the megaplasmids and chromosomal constructs were very similar, and reinforced the view that highest expression in Synechocystis can be obtained using RSF1010-derived replicative vectors or the native small plasmid pCA2.4 evaluated in comparison. Parallel replicon copy number analysis by RT-qPCR showed that the expression from the alternative loci is largely determined by the gene dosage in Synechocystis, thereby confirming the dependence formerly proposed based on literature. CONCLUSIONS: This study brings together nine different integrative loci in the genome of Synechocystis to demonstrate quantitative differences between target sites in the chromosome, the native plasmids, and a RSF1010-based replicative expression vector. To date, this is the most comprehensive comparison of alternative integrative sites in Synechocystis, and provides the first direct reference between expression efficiency and replicon gene dosage in the context. In the light of existing literature, the findings support the view that the small native plasmids can be notably more difficult to target than the chromosome or the megaplasmids, and that the RSF1010-derived vectors may be surprisingly well maintained under non-selective culture conditions in this cyanobacterial host. Altogether, the work broadens our views on genomic integration and the rational use of different integrative loci versus replicative plasmids, when aiming at expressing heterologous genes in Synechocystis.

Heterologous Production of Glycine Betaine Using Synechocystis sp. PCC 6803-Based Chassis Lacking Native Compatible Solutes.

Among compatible solutes, glycine betaine has various applications in the fields of nutrition, pharmaceuticals, and cosmetics. Currently, this compound can be extracted from sugar beet plants or obtained by chemical synthesis, resulting in low yields or high carbon footprint, respectively. Hence, in this work we aimed at exploring the production of glycine betaine using the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a photoautotrophic chassis. Synechocystis mutants lacking the native compatible solutes sucrose or/and glucosylglycerol-∆sps, ∆ggpS, and ∆sps∆ggpS-were generated and characterized. Under salt stress conditions, the growth was impaired and accumulation of glycogen decreased by ∼50% whereas the production of compatible solutes and extracellular polymeric substances (capsular and released ones) increased with salinity. These mutants were used as chassis for the implementation of a synthetic device based on the metabolic pathway described for the halophilic cyanobacterium Aphanothece halophytica for the production of the compatible solute glycine betaine. Transcription of ORFs comprising the device was shown to be stable and insulated from Synechocystis' native regulatory network. Production of glycine betaine was achieved in all chassis tested, and was shown to increase with salinity. The introduction of the glycine betaine synthetic device into the ∆ggpS background improved its growth and enabled survival under 5% NaCl, which was not observed in the absence of the device. The maximum glycine betaine production [64.29 µmol/gDW (1.89 µmol/mg protein)] was reached in the ∆ggpS chassis grown under 3% NaCl. Taking into consideration this production under seawater-like salinity, and the identification of main key players involved in the carbon fluxes, this work paves the way for a feasible production of this, or other compatible solutes, using optimized Synechocystis chassis in a pilot-scale.

Modulation of Intracellular O2 Concentration in Escherichia coli Strains Using Oxygen Consuming Devices.

The use of cell factories for the production of bulk and value-added compounds is nowadays an advantageous alternative to the traditional petrochemical methods. Nevertheless, the efficiency and productivity of several of these processes can improve with the implementation of micro-oxic or anoxic conditions. In the industrial setting, laccases are appealing catalysts that can oxidize a wide range of substrates and reduce O2 to H2O. In this work, several laccase-based devices were designed and constructed to modulate the intracellular oxygen concentration in bacterial chassis. These oxygen consuming devices (OCDs) included Escherichia coli's native laccase (CueO) and three variants of this protein obtained by directed evolution. The OCDs were initially characterized in vitro using E. coli DH5α protein extracts and subsequently using extracts obtained from other E. coli strains and in vivo. Upon induction of the OCDs, no major effect on growth was observed in four of the strains tested, and analysis of the cell extract protein profiles revealed increased levels of laccase. Moreover, oxygen consumption associated with the OCDs occurred under all of the conditions tested, but the performance of the devices was shown to be strain-dependent, highlighting the importance of the genetic background even in closely related strains. One of the laccase variants showed 13- and 5-fold increases in oxidase activity and O2 consumption rate, respectively. Furthermore, it was also possible to demonstrate O2 consumption in vivo using l-DOPA as the substrate, which represents a proof of concept that these OCDs generate an intracellular oxygen sink, thereby manipulating the redox status of the cells. In addition, the modularity and orthogonality principles used for the development of these devices allow easy reassembly and fine-tuning, foreseeing their introduction into other chassis/systems.

Identification of inner membrane translocase components of TolC-mediated secretion in the cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria were the first organisms ever to perform oxygenic photosynthesis and still significantly contribute to primary production on a global scale. To assure the proper functioning of their primary metabolism and cell homeostasis, cyanobacteria must rely on efficient transport systems to cross their multilayered cell envelope. However, cyanobacterial secretion mechanisms remain largely unknown. Here, we report on the identification of 11 putative inner membrane translocase components of TolC-mediated secretion in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Gene-inactivation of each of the candidate genes followed by a comprehensive phenotypic characterization allowed to link specific protein components to the processes of protein export (as part of the type I secretion system) and drug efflux (part of the resistance-division-nodulation efflux pumps). In addition, mutants in genes sll0141, sll0180 and slr0369 exhibited alterations in pilin glycosylation, but pili structures could still be observed by transmission electron microscopy. By studying the release of outer membrane vesicles (OMVs), an alternative secretion route, on mutants with impaired secretory functions we suggest that the hyper-vesiculating phenotype of the TolC-deficient mutant is related to cell envelope stress management. Altogether, these findings highlight how both classical (TolC-mediated) and nonclassical (OMVs-mediated) secretion systems are crucial for cyanobacterial cell homeostasis.

Expanding the toolbox for Synechocystis sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters.

Cyanobacteria are promising 'low-cost' cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the 'green' Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference P rnpB , three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study.

Improving a Synechocystis-based photoautotrophic chassis through systematic genome mapping and validation of neutral sites.

The use of microorganisms as cell factories frequently requires extensive molecular manipulation. Therefore, the identification of genomic neutral sites for the stable integration of ectopic DNA is required to ensure a successful outcome. Here we describe the genome mapping and validation of five neutral sites in the chromosome of Synechocystis sp. PCC 6803, foreseeing the use of this cyanobacterium as a photoautotrophic chassis. To evaluate the neutrality of these loci, insertion/deletion mutants were produced, and to assess their functionality, a synthetic green fluorescent reporter module was introduced. The constructed integrative vectors include a BioBrick-compatible multiple cloning site insulated by transcription terminators, constituting robust cloning interfaces for synthetic biology approaches. Moreover, Synechocystis mutants (chassis) ready to receive purpose-built synthetic modules/circuits are also available. This work presents a systematic approach to map and validate chromosomal neutral sites in cyanobacteria, and that can be extended to other organisms.

HesF, an exoprotein required for filament adhesion and aggregation in Anabaena sp. PCC 7120.

Here, we report on the identification and characterization of a protein (Alr0267) named HesF, found in the extracellular milieu of Anabaena sp. PCC 7120 grown diazotrophically. hesF was found to be highly upregulated upon transition from non-nitrogen-fixing to nitrogen-fixing conditions, and the highest transcript levels were detected towards the end of the heterocyst differentiation process. The hesF promoter drives transcription of the gene in heterocysts only, and both NtcA and HetR are essential for the gene's in vivo activation. An examination of HesF's translocation showed that the secretion system is neither heterocyst-specific nor dependent on nitrogen-fixing conditions. Furthermore, HesF was found to be a type I secretion system substrate, since an HgdD mutant failed to secrete HesF. Several analyses revealed that a HesF minus mutant strain lacks the heterocyst-specific polysaccharide fibrous layer, accumulates high amounts of polysaccharides in the medium and that HesF is essential for the typical aggregation phenotype in diazotrophic conditions. Thus, we propose that HesF is a carbohydrate-binding exoprotein that plays a role in maintaining the heterocyst cell wall structure. A combination of and possibly interaction between HesF and heterocyst-specific polysaccharides seems to be responsible for filament adhesion and culture aggregation in heterocyst-forming cyanobacteria.

Selection of suitable reference genes for RT-qPCR analyses in cyanobacteria.

Cyanobacteria are a group of photosynthetic prokaryotes that have a diverse morphology, minimal nutritional requirements and metabolic plasticity that has made them attractive organisms to use in biotechnological applications. The use of these organisms as cell factories requires the knowledge of their physiology and metabolism at a systems level. For the quantification of gene transcripts real-time quantitative polymerase chain reaction (RT-qPCR) is the standard technique. However, to obtain reliable RT-qPCR results the use and validation of reference genes is mandatory. Towards this goal we have selected and analyzed twelve candidate reference genes from three morphologically distinct cyanobacteria grown under routinely used laboratory conditions. The six genes exhibiting less variation in each organism were evaluated in terms of their expression stability using geNorm, NormFinder and BestKeeper. In addition, the minimum number of reference genes required for normalization was determined. Based on the three algorithms, we provide a list of genes for cyanobacterial RT-qPCR data normalization. To our knowledge, this is the first work on the validation of reference genes for cyanobacteria constituting a valuable starting point for future works.

Construction of a chassis for hydrogen production: physiological and molecular characterization of a Synechocystis sp. PCC 6803 mutant lacking a functional bidirectional hydrogenase.

Cyanobacteria are photosynthetic prokaryotes that are promising 'low-cost' microbial cell factories due to their simple nutritional requirements and metabolic plasticity, and the availability of tools for their genetic manipulation. The unicellular non-nitrogen-fixing Synechocystis sp. PCC 6803 is the best studied cyanobacterial strain and its genome was the first to be sequenced. The vast amount of physiological and molecular data available, together with a relatively small genome, makes Synechocystis suitable for computational metabolic modelling and to be used as a photoautotrophic chassis in synthetic biology applications. To prepare it for the introduction of a synthetic hydrogen producing device, a Synechocystis sp. PCC 6803 deletion mutant lacking an active bidirectional hydrogenase (ΔhoxYH) was produced and characterized at different levels: physiological, proteomic and transcriptional. The results showed that, under conditions favouring hydrogenase activity, 17 of the 210 identified proteins had significant differential fold changes in comparisons of the mutant with the wild-type. Most of these proteins are related to the redox and energy state of the cell. Transcriptional studies revealed that only six genes encoding those proteins exhibited significant differences in transcript levels. Moreover, the mutant exhibits similar growth behaviour compared with the wild-type, reflecting Synechocystis plasticity and metabolic adaptability. Overall, this study reveals that the Synechocystis ΔhoxYH mutant is robust and can be used as a photoautotrophic chassis for the integration of synthetic constructs, i.e. molecular constructs assembled from well characterized biological and/or synthetic parts (e.g. promoters, regulators, coding regions, terminators) designed for a specific purpose.

Infection levels and diversity of anisakid nematodes in blackspot seabream, Pagellus bogaraveo, from Portuguese waters.

The blackspot seabream, Pagellus bogaraveo, is a sparid fish of great economic importance in the northeast Atlantic. The main aim of this work was to assess the infection levels and diversity of anisakid nematodes parasitizing P. bogaraveo from Portuguese waters. The anisakid larvae were identified by polymerase chain reaction-restriction fragment length polymorphism analysis and ten different patterns were observed, four of which were not previously reported in the literature. Moreover, several species were detected for the first time in this host: Anisakis simplex × Anisakis pegreffii hybrids, Anisakis ziphidarum, Anisakis typica, Anisakis physeteris, as well as three undescribed anisakids Anisakis sp. PB-2009, Anisakis sp. PB-2010, and Contracaecum sp. PB-2010. The ITS1-5.8S-ITS2 region was sequenced and analyzed phylogenetically, revealing that our anisakids were distributed by the two distinct clades reported previously, corresponding to the two recognized larval morphotypes. Moreover, a group of organisms, including our specimens from Madeira and the previously reported Anisakis sp. HC-2005, cluster together and seem to belong to clade I. A certain degree of intraspecific diversity was also detected. Samples from mainland waters had the highest infection levels and were dominated by A. pegreffii. Madeira had the highest diversity overall, dominated by Anisakis sp. PB-2010. Fish from the Azores had the lowest infection levels, and the species with the highest relative abundance was A. physeteris. The anisakid nematode communities were relatively similar in mainland waters but very distinct in both the Azores and Madeira islands, suggesting the existence of at least three different stocks of P. bogaraveo in the northeast Atlantic.

Labrys portucalensis sp. nov., a fluorobenzene-degrading bacterium isolated from an industrially contaminated sediment in northern Portugal.

A detailed classification of a novel bacterial strain, designated F11(T), capable of degrading fluorobenzene as a sole carbon and energy source, was performed by using a polyphasic approach. This Gram-negative, rod-shaped, non-motile, non-spore-forming, aerobic bacterium was isolated from a sediment sample collected from an industrially contaminated site in northern Portugal. The predominant whole-cell fatty acids were C(19 : 0) cyclo omega8c, C(16 : 0), C(18 : 1)omega7c, C(18 : 0), C(18 : 0) 3-OH and C(16 : 0) 3-OH. The G+C content of the DNA was 62.9 mol% and the major respiratory quinone was ubiquinone 10 (UQ-10). 16S rRNA gene sequence analysis revealed that strain F11(T) was a member of the class Alphaproteobacteria and was phylogenetically related to the genus Labrys, having sequence similarities of 95.6 and 93.1 % to the type strains of Labrys monachus and Labrys methylaminiphilus, respectively. DNA-DNA hybridization experiments revealed levels of relatedness of <70 % between strain F11(T) and the type strains of L. monachus and L. methylaminiphilus (38.6 and 34.1 %, respectively), justifying the classification of strain F11(T) as representing a novel species of the genus Labrys. The name Labrys portucalensis sp. nov. is proposed for this organism. The type strain is F11(T) (=LMG 23412(T)=DSM 17916(T)).

Role of respiration and glutathione in cadmium-induced oxidative stress in Escherichia coli K-12.

Cadmium is a widespread pollutant that has been associated with oxidative stress, but the mechanism behind this effect in prokaryotes is still unclear. In this work, we exposed two glutathione deficient mutants (DeltagshA and DeltagshB) and one respiration deficient mutant (DeltaubiE) to a sublethal concentration of cadmium. The glutathione mutants show a similar increase in reactive oxygen species as the wild type. Experiments performed using the DeltaubiE strain showed that this mutant is more resistant to cadmium ions and that Cd-induced reactive oxygen species levels were not altered. In the light of these facts, we conclude that the interference of cadmium with the respiratory chain is the cause of the oxidative stress induced by this metal and that, contrary to previously proposed models, the reactive oxygen species increase is not due to glutathione depletion, although this peptide is crucial for cadmium detoxification.

Methyloversatilis universalis gen. nov., sp. nov., a novel taxon within the Betaproteobacteria represented by three methylotrophic isolates.

The taxonomic positions and phylogenetic relationships of two new methylotrophic isolates from Lake Washington (USA) sediment, FAM5T and 500, and the previously described methylotrophic strain EHg5 isolated from contaminated soil in Estarreja (Portugal) were investigated. All three strains were facultative methylotrophs capable of growth on a variety of C1 and multicarbon compounds. Optimal growth occurred at pH 7.5-8 and 30-37 degrees C. The major fatty acids were C16:1omega7c and C16:0. The major quinone was ubiquinone Q8. Neither methanol dehydrogenase nor methanol oxidase activities were detectable in cells grown on methanol, suggesting an alternative, as-yet unknown, mechanism for methanol oxidation. The isolates assimilated C1 units at the level of formaldehyde, via the serine cycle. The DNA G+C content of the strains ranged between 64 and 65 mol%. 16S rRNA gene sequence similarity between the three new isolates was 99.85-100%, but was below 94% with other members of the Betaproteobacteria, indicating that the isolates represent a novel taxon. Based on physiological, phenotypic and genomic characteristics of the three isolates, a new genus, Methyloversatilis gen. nov., is proposed within the family Rhodocyclaceae. The type strain of Methyloversatilis universalis gen. nov., sp. nov. is FAM5T (=CCUG 52030T=JCM 13912T).

Identification, mutagenesis, and transcriptional analysis of the methanesulfonate transport operon of Methylosulfonomonas methylovora.

Recently identified genes located downstream (3') of the msmEF (transport encoding) gene cluster, msmGH, and located 5' of the structural genes for methanesulfonate monooxygenase (MSAMO) are described from Methylosulfonomonas methylovora. Sequence analysis of the derived polypeptide sequences encoded by these genes revealed a high degree of identity to ABC-type transporters. MsmE showed similarity to a putative periplasmic substrate binding protein, MsmF resembled an integral membrane-associated protein, and MsmG was a putative ATP-binding enzyme. MsmH was thought to be the cognate permease component of the sulfonate transport system. The close association of these putative transport genes to the MSAMO structural genes msmABCD suggested a role for these genes in transport of methanesulfonic acid (MSA) into M. methylovora. msmEFGH and msmABCD constituted two operons for the coordinated expression of MSAMO and the MSA transporter systems. Reverse-transcription-PCR analysis of msmABCD and msmEFGH revealed differential expression of these genes during growth on MSA and methanol. The msmEFGH operon was constitutively expressed, whereas MSA induced expression of msmABCD. A mutant defective in msmE had considerably slower growth rates than the wild type, thus supporting the proposed role of MsmE in the transport of MSA into M. methylovora.

Isolation and properties of methanesulfonate-degrading Afipia felis from Antarctica and comparison with other strains of A. felis.

Three novel strains of methylotrophic Afipia felis were isolated from several locations on Signy Island, Antarctica, and a fourth from estuary sediment from the River Douro, Portugal. They were identified as strains of the alpha-2 proteobacterium A. felis by 16S rRNA gene sequence analysis. Two strains tested were shown to contain the fdxA gene, diagnostic for A. felis. All strains grew with methanesulfonate (and two strains with dimethylsulfone) as sole carbon substrate. Growth on methanesulfonate required methanesulfonate monooxygenase (MSAMO), using NADH as the reductant and stimulated by reduced flavin nucleotides and Fe(II). Polymerase chain reaction amplification of DNA from an Antarctic strain showed a typical msmA gene for the alpha-hydroxylase of MSAMO, and both Antarctic and Portuguese strains contained mxaF, the methanol dehydrogenase large subunit gene. This is the first report of methanesulfonate-degrading bacteria from the Antarctic and of methylotrophy in Afipia, and the first description of any bacterium able to use both methanesulfonate and dimethylsulfone. In contrast, the type strain of A. felis DSM 7326(T) was not methylotrophic, but grew in defined mineral medium with a wide range of single simple organic substrates. Free-living Afipia strains occurring widely in the natural environment may be significant as methylotrophs, degrading C(1)-sulfur compounds, including the recalcitrant organosulfur compound methanesulfonate.

Novel pollutant-resistant methylotrophic bacteria for use in bioremediation.

Thirty one novel methylotrophic bacterial strains were isolated from a range of soil and sediment sources (both pristine and polluted) under different enrichment regimes. They were characterised physiologically and classified by their 16S rRNA gene sequence. A great taxonomical and phenotypical variety was recovered. Some of the isolates display interesting features of resistance to heavy metals, arsenate or organic pollution and four can be considered real 'super-bugs' for their ability to withstand extremely high concentrations of a variety of pollutants. A description of the 31 strains is presented in this work.