Paolo De Marco

Methylosulfonomonas methylovora gen. nov., sp. nov., and Marinosulfonomonas methylotropha gen. nov., sp. nov.: novel methylotrophs able to grow on methanesulfonic acid

Abstract Two novel genera of restricted facultative methylotrophs are described; both Methylosulfonomonas and Marinosulfonomonas are unique in being able to grow on methanesulfonic acid as their sole source of carbon and energy. Five identical strains of Methylosulfonomonas were isolated from diverse soil samples in England and were shown to differ in their morphology, physiology, DNA base composition, molecular genetics, and 16S rDNA sequences from the two marine strains of Marinosulfonomonas, which were isolated from British coastal waters. The marine strains were almost indistinguishable from each other and are considered to be strains of one species. Type species of each genus have been identified and named Methylosulfonomonas methylovora (strain M2) and Marinosulfonomonas methylotropha (strain PSCH4). Phylogenetic analysis using 16S rDNA sequencing places both genera in the α-Proteobacteria. Methylosulfonomonas is a discrete lineage within the α-2 subgroup and is not related closely to any other known bacterial genus. The Marinosulfonomonas strains form a monophyletic cluster in the α-3 subgroup of the Proteobacteria with Roseobacter spp. and some other partially characterized marine bacteria, but they are distinct from these at the genus level. This work shows that the isolation of bacteria with a unique biochemical character, the ability to grow on methanesulfonic acid as energy and carbon substrate, has resulted in the identification of two novel genera of methylotrophs that are unrelated to any other extant methylotroph genera.

Heterologous expression of soluble methane monooxygenase genes in methanotrophs containing only particulate methane monooxygenase

Abstract The methanotrophs Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b contain particulate methane monooxygenase (pMMO) and soluble methane monooxygenase (sMMO) genes. Other methanotrophs such as Methylomicrobium album BG8 and Methylocystis parvus OBBP contain only pMMO genes. Although molecular genetic techniques are poorly developed in methanotrophs, sMMO genes were expressed in methanotrophs normally containing only pMMO genes. This was achieved by conjugation using broad-host-range plasmids containing the native promoter and sMMO genes from Mc. capsulatus (Bath) and Ms. trichosporium OB3b. sMMO genes derived from Ms. trichosporium OB3b were expressed in an active form in Mcy. parvus OBBP and in Mm. album BG8. Therefore, all of the genes required for active sMMO synthesis were contained on the broad-host-range plasmids and were expressed in the heterologous hosts. Constitutive synthesis of pMMO was observed in Mm. album BG8 when grown at high and low copper-to-biomass ratios, while transcription of the recombinant sMMO genes was only observed under growth conditions of low copper-to-biomass ratios. Therefore, the regulatory protein(s) for sMMO synthesis was also present on the plasmid used, or the heterologous host contained a regulatory system for sMMO. Expression of sMMO genes in methanotrophs containing only pMMO will assist further investigations on the expression and regulation of MMO genes in methanotrophs.

Duplicate copies of genes encoding methanesulfonate monooxygenase in Marinosulfonomonas methylotropha strain TR3 and detection of methanesulfonate utilizers in the environment.

ABSTRACT Marinosulfonomonas methylotropha strain TR3 is a marine methylotroph that uses methanesulfonic acid (MSA) as a sole carbon and energy source. The genes from M. methylotropha strain TR3 encoding methanesulfonate monooxygenase, the enzyme responsible for the initial oxidation of MSA to formaldehyde and sulfite, were cloned and sequenced. They were located on two gene clusters on the chromosome of this bacterium. A 5.0-kbp HindIII fragment contained msmA, msmB, and msmC, encoding the large and small subunits of the hydroxylase component and the ferredoxin component, respectively, of the methanesulfonate monooxygenase, while a 6.5-kbp HindIII fragment contained duplicate copies of msmA and msmB, as well as msmD, encoding the reductase component of methanesulfonate. Both sets of msmA and msmB genes were virtually identical, and the derived msmA and msmB sequences of M. methylotropha strain TR3, compared with the corresponding hydroxylase from the terrestrial MSA utilizer Methylosulfonomonas methylovora strain M2 were found to be 82 and 69% identical. The msmA gene was investigated as a functional gene probe for detection of MSA-utilizing bacteria. PCR primers spanning a region of msmA which encoded a unique Rieske [2Fe-2S] binding region were designed. These primers were used to amplify the corresponding msmA genes from newly isolated Hyphomicrobium, Methylobacterium, and Pedomicrobium species that utilized MSA, from MSA enrichment cultures, and from DNA samples extracted directly from the environment. The high degree of identity of these msmA gene fragments, compared to msmA sequences from extant MSA utilizers, indicated the effectiveness of these PCR primers in molecular microbial ecology.

Enrichment of microbial cultures able to degrade 1,3-dichloro-2-propanol : a comparison between batch and continuous methods

Microbial cultures able to degrade xenobiotic compounds are the key element for biological treatment of waste effluents and are obtained from enrichment processes. In this study, two common enrichment methods, suspension batch and immobilized continuous, were compared. The main selection factor was the presence of 1,3-dichloro-2-propanol (1,3-DCP) as the single carbon source. Both methods have successfully enriched microbial consortia able to degrade 1,3-DCP. When tested in batch culture, the degradation rates of 1,3-DCP by the two consortia were different, with the consortia obtained by batch enrichment presenting slightly higher rates. A preliminary morphological and biochemical analysis of the predominant colonial types present in each degrading consortia revealed the presence of different constituting strains. Three bacterial isolates capable of degrading 1,3-DCP as single strains were obtained from the batch enrichments. These strains were classified by 16S rRNA analysis as belonging to the Rhizobiaceae group. Degradation rates of 1,3-DCP were lower when single species were used, reaching 45 mg l-1 d-1, as compared to 74 mg l-1 d-1 of the consortia enriched on the batch method. Mutualistic interactions may explain the better performance of the enriched consortia.

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 (ΔgshA and ΔgshB) and one respiration deficient mutant (ΔubiE) 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 ΔubiE 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.

Chryseobacterium palustre sp. nov. and Chryseobacterium humi sp. nov., isolated from industrially contaminated sediments

Two Gram-staining-negative bacterial strains, designated 3A10(T) and ECP37(T), were isolated from sediment samples collected from an industrially contaminated site in northern Portugal. These two organisms were rod-shaped, non-motile, aerobic, catalase- and oxidase-positive and formed yellow colonies. The predominant fatty acids were iso-C(15 : 0), anteiso-C(15 : 0), iso-C(17 : 1)omega9c and iso-C(17 : 0) 3-OH. The G+C content of the DNA of strains 3A10(T) and ECP37(T) was 43 and 34 mol%, respectively. The major isoprenoid quinone of the two strains was MK-6. 16S rRNA gene sequence analysis revealed that strains 3A10(T) and ECP37(T) were members of the family Flavobacteriaceae and were related phylogenetically to the genus Chryseobacterium. Strain 3A10(T) showed 16S rRNA gene sequence similarity values of 97.2 and 96.6 % to the type strains of Chryseobacterium antarcticum and Chryseobacterium jeonii, respectively; strain ECP37(T) showed 97.3 % similarity to the type strain of Chryseobacterium marinum. DNA-DNA hybridization experiments revealed levels of genomic relatedness of <70 % between strains 3A10(T) and ECP37(T) and between these two strains and the type strains of C. marinum, C. antarcticum and C. jeonii, justifying their classification as representing two novel species of the genus Chryseobacterium. The names proposed for these organisms are Chryseobacterium palustre sp. nov. (type strain 3A10(T) =LMG 24685(T) =NBRC 104928(T)) and Chryseobacterium humi sp. nov. (type strain ECP37(T) =LMG 24684(T) =NBRC 104927(T)).

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)).

Isolation and characterization of two new methanesulfonic acid-degrading bacterial isolates from a Portuguese soil sample

Abstract Two novel bacterial strains that can utilize methanesulfonic acid as a source of carbon and energy were isolated from a soil sample collected in northern Portugal. Morphological, physiological, biochemical and molecular biological characterization of the two isolates indicate that strain P1 is a pink-pigmented facultative methylotroph belonging to the genus Methylobacterium, while strain P2 is a restricted methylotroph belonging to the genus Hyphomicrobium. Both strains are strictly aerobic, degrade methanesulfonate, and release small quantities of sulfite into the medium. Growth on methanesulfonate induces a specific polypeptide profile in each strain. This, together with the positive hybridization to a DNA probe that carries the msm genes of Methylosulfonomonas methylovora strain M2, strongly endorses the contention that a methanesulfonic acid monooxygenase related to that found in the previously known methanesulfonate-utilizing bacteria is present in strains P1 and P2. The isolation of bacteria containing conserved msm genes from diverse environments and geographical locations supports the hypothesis that a common enzyme may be globally responsible for the oxidation of methanesulfonate by natural methylotrophic communities.

Functional and genomic diversity of methylotrophic Rhodocyclaceae: description of Methyloversatilis discipulorum sp. nov.

Three strains of methylotrophic Rhodocyclaceae (FAM1(T), RZ18-153 and RZ94) isolated from Lake Washington sediment samples were characterized. Based on phylogenetic analysis of 16S rRNA gene sequences the strains should be assigned to the genus Methyloversatilis. Similarly to other members of the family, the strains show broad metabolic capabilities and are able to utilize a number of organic acids, alcohols and aromatic compounds in addition to methanol and methylamine. The main fatty acids were 16:1ω7c (49-59%) and 16:0 (32-29%). Genomes of all isolates were sequenced, assembled and annotated in collaboration with the DOE Joint Genome Institute (JGI). Genome comparison revealed that the strains FAM1T, RZ18-153 and RZ94 are closely related to each other and almost equally distant from two previously described species of the genus Methyloversatilis, Methyloversatilis universalis and Methyloversatilis thermotolerans. Like other methylotrophic species of the genus Methyloversatilis, all three strains possess one-subunit PQQ-dependent ethanol/methanol dehydrogenase (Mdh-2), the N-methylglutamate pathway and the serine cycle (isocitrate lyase/malate synthase, Icl/ms(+) variant). Like M. universalis, strains FAM1(T), RZ18-153 and RZ94 have a quinohemoprotein amine dehydrogenase, a tungsten-containing formaldehyde ferredoxin oxidoreductase, phenol hydroxylase, and the complete Calvin cycle. Similarly to M. thermotolerans, the three strains possess two-subunit methanol dehydrogenase (MxaFI), monoamine oxidase (MAO) and nitrogenase. Based on the phenotypic and genomic data, the strains FAM1(T), RZ18-153 and RZ94 represent a novel species of the genus Methyloversatilis, for which the name Methyloversatilis discipulorum sp. nov. is proposed. The type strain is FAM1(T) ( = JCM 30542(T) = VKM = B-2888(T)).

Functional and genomic diversity of methylotrophic Rhodocyclaceae: description of the new species Methyloversatilis discipulorum sp. nov.

Three strains of methylotrophic Rhodocyclaceae (FAM1(T), RZ18-153 and RZ94) isolated from Lake Washington sediment samples were characterized. Based on phylogenetic analysis of 16S rRNA gene sequences the strains should be assigned to the genus Methyloversatilis. Similarly to other members of the family, the strains show broad metabolic capabilities and are able to utilize a number of organic acids, alcohols and aromatic compounds in addition to methanol and methylamine. The main fatty acids were 16:1ω7c (49-59%) and 16:0 (32-29%). Genomes of all isolates were sequenced, assembled and annotated in collaboration with the DOE Joint Genome Institute (JGI). Genome comparison revealed that the strains FAM1T, RZ18-153 and RZ94 are closely related to each other and almost equally distant from two previously described species of the genus Methyloversatilis, Methyloversatilis universalis and Methyloversatilis thermotolerans. Like other methylotrophic species of the genus Methyloversatilis, all three strains possess one-subunit PQQ-dependent ethanol/methanol dehydrogenase (Mdh-2), the N-methylglutamate pathway and the serine cycle (isocitrate lyase/malate synthase, Icl/ms(+) variant). Like M. universalis, strains FAM1(T), RZ18-153 and RZ94 have a quinohemoprotein amine dehydrogenase, a tungsten-containing formaldehyde ferredoxin oxidoreductase, phenol hydroxylase, and the complete Calvin cycle. Similarly to M. thermotolerans, the three strains possess two-subunit methanol dehydrogenase (MxaFI), monoamine oxidase (MAO) and nitrogenase. Based on the phenotypic and genomic data, the strains FAM1(T), RZ18-153 and RZ94 represent a novel species of the genus Methyloversatilis, for which the name Methyloversatilis discipulorum sp. nov. is proposed. The type strain is FAM1(T) ( = JCM 30542(T) = VKM = B-2888(T)).