Genes of the N-methylglutamate pathway are essential for growth of Methylobacterium extorquens DM4 with monomethylamine.

Monomethylamine (MMA, CH3NH2) can be used as a carbon and nitrogen source by many methylotrophic bacteria. Methylobacterium extorquens DM4 lacks the MMA dehydrogenase encoded by mau genes, which in M. extorquens AM1 is essential for growth on MMA. Identification and characterization of minitransposon mutants with an MMA-dependent phenotype showed that strain DM4 grows with MMA as the sole source of carbon, energy, and nitrogen by the N-methylglutamate (NMG) pathway. Independent mutations were found in a chromosomal region containing the genes gmaS, mgsABC, and mgdABCD for the three enzymes of the pathway, γ-glutamylmethylamide (GMA) synthetase, NMG synthase, and NMG dehydrogenase, respectively. Reverse transcription-PCR confirmed the operonic structure of the two divergent gene clusters mgsABC-gmaS and mgdABCD and their induction during growth with MMA. The genes mgdABCD and mgsABC were found to be essential for utilization of MMA as a carbon and nitrogen source. The gene gmaS was essential for MMA utilization as a carbon source, but residual growth of mutant DM4gmaS growing with succinate and MMA as a nitrogen source was observed. Plasmid copies of gmaS and the gmaS homolog METDI4690, which encodes a protein 39% identical to GMA synthetase, fully restored the ability of mutants DM4gmaS and DM4gmaSΔmetdi4690 to use MMA as a carbon and nitrogen source. Similarly, chemically synthesized GMA, the product of GMA synthetase, could be used as a nitrogen source for growth in the wild-type strain, as well as in DM4gmaS and DM4gmaSΔmetdi4690 mutants. The NADH:ubiquinone oxidoreductase respiratory complex component NuoG was also found to be essential for growth with MMA as a carbon source.

GTPase Era at the heart of ribosome assembly.

Ribosome biogenesis is a key process in all organisms. It relies on coordinated work of multiple proteins and RNAs, including an array of assembly factors. Among them, the GTPase Era stands out as an especially deeply conserved protein, critically required for the assembly of bacterial-type ribosomes from Escherichia coli to humans. In this review, we bring together and critically analyze a wealth of phylogenetic, biochemical, structural, genetic and physiological data about this extensively studied but still insufficiently understood factor. We do so using a comparative and, wherever possible, synthetic approach, by confronting observations from diverse groups of bacteria and eukaryotic organelles (mitochondria and chloroplasts). The emerging consensus posits that Era intervenes relatively early in the small subunit biogenesis and is essential for the proper shaping of the platform which, in its turn, is a prerequisite for efficient translation. The timing of Era action on the ribosome is defined by its interactions with guanosine nucleotides [GTP, GDP, (p)ppGpp], ribosomal RNA, and likely other factors that trigger or delay its GTPase activity. As a critical nexus of the small subunit biogenesis, Era is subject to sophisticated regulatory mechanisms at the transcriptional, post-transcriptional, and post-translational levels. Failure of these mechanisms or a deficiency in Era function entail dramatic generalized consequences for the protein synthesis and far-reaching, pleiotropic effects on the organism physiology, such as the Perrault syndrome in humans.

Complete genome sequences of six strains of the genus Methylobacterium.

The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.

A Methylotrophic Bacterium Growing with the Antidiabetic Drug Metformin as Its Sole Carbon, Nitrogen and Energy Source.

Metformin is one of the most prescribed antidiabetic agents worldwide and is also considered for other therapeutic applications including cancer and endocrine disorders. It is largely unmetabolized by human enzymes and its presence in the environment has raised concern, with reported toxic effects on aquatic life and potentially also on humans. We report on the isolation and characterisation of strain MD1, an aerobic methylotrophic bacterium growing with metformin as its sole carbon, nitrogen and energy source. Strain MD1 degrades metformin into dimethylamine used for growth, and guanylurea as a side-product. Sequence analysis of its fully assembled genome showed its affiliation to Aminobacter niigataensis. Differential proteomics and transcriptomics, as well as mini-transposon mutagenesis of the strain, point to genes and proteins essential for growth with metformin and potentially associated with hydrolytic C-N cleavage of metformin or with cellular transport of metformin and guanylurea. The obtained results suggest the recent evolution of the growth-supporting capacity of strain MD1 to degrade metformin. Our results identify candidate proteins of the enzymatic system for metformin transformation in strain MD1 and will inform future research on the fate of metformin and its degradation products in the environment and in humans.

Complete genome sequence of the chloromethane-degrading Hyphomicrobium sp. strain MC1.

Hyphomicrobium sp. strain MC1 is an aerobic methylotroph originally isolated from industrial sewage. This prosthecate bacterium was the first strain reported to grow with chloromethane as the sole carbon and energy source. Its genome, consisting of a single 4.76-Mb chromosome, is the first for a chloromethane-degrading bacterium to be formally reported.

FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter.

The first step in the specific uptake of iron via siderophores in Gram-negative bacteria is the recognition and binding of a ferric siderophore by its cognate receptor. We investigated the molecular basis of this event through structural and biochemical approaches. FpvA, the pyoverdine-Fe transporter from Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is able to transport ferric-pyoverdines originating from other species, whereas most fluorescent pseudomonads are only able to use the one they produce among the more than 100 known different pyoverdines. We solved the structure of FpvA bound to non-cognate pyoverdines of high- or low-affinity and found a close correlation between receptor-ligand structure and the measured affinities. The structure of the first amino acid residues of the pyoverdine chain distinguished the high- and low-affinity binders while the C-terminal portion of the pyoverdines, often cyclic, does not appear to contribute extensively to the interaction between the siderophore and its transporter. The specificity of the ferric-pyoverdine binding site of FpvA is conferred by the structural elements common to all ferric-pyoverdines, i.e. the chromophore, iron, and its chelating groups.

Coupling of denaturing high-performance liquid chromatography and terminal restriction fragment length polymorphism with precise fragment sizing for microbial community profiling and characterization.

Terminal restriction fragment length polymorphism (T-RFLP) is used to monitor the structural diversity of complex microbial communities in terms of richness, relative abundance, and distribution of the major subpopulations and individual members. However, discrepancies of several nucleotides between expected and experimentally observed lengths of terminal restriction fragments (T-RFs), together with the difficulty of obtaining DNA sequence information from T-RFLP profiling, often prevent accurate phylogenetic characterization of the microbial community of interest. In this study, T-RFLP analysis of DNA from an artificial assembly of five bacterial strains was carried out with a combination of two size markers with different fluorescent tags. Precise sizing of T-RFs in the 50- to 500-nucleotide range was achieved by using the same dye for both samples and size markers. Phylogenetic assignment of the component microbial strains was facilitated by coupling T-RFLP to denaturing high-performance liquid chromatography (D-HPLC) of 16S RNA gene fragments followed by direct sequencing. The proposed coupling of D-HPLC and T-RFLP provides unambiguous characterization of microbial communities containing less than 15 microbial strains.

Genome Sequence of the Dichloromethane-Degrading Bacterium Hyphomicrobium sp. Strain GJ21.

The genome sequence of Hyphomicrobium sp. strain GJ21, isolated in the Netherlands from samples of environments contaminated with halogenated pollutants and capable of using dichloromethane as its sole carbon and energy source, was determined.

Tetrachloromethane-Degrading Bacterial Enrichment Cultures and Isolates from a Contaminated Aquifer.

The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl4) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L(-1) CCl4, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl4 degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl4 transformation. As estimated by T-RFLP, phylotypes of CCl4-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community.

Pyoverdine synthesis by the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1.

When iron-starved, the Mn(II)-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB-1 and PVDMnB1), siderophores that both influence iron uptake and inhibit manganese(II) oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS, and various siderotyping techniques were used to identify the peptides of PVDGB-1 and PVDMnB1 as being (for both PVDs): chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase (NRPS), coded by the operon PputGB1_4083-4086, could produce the peptide backbone of PVDGB-1. To verify this prediction, plasmid integration disruption of PputGB1_4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1_4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II)-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group, and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains) were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III).

Methylobacterium genome sequences: a reference blueprint to investigate microbial metabolism of C1 compounds from natural and industrial sources.

BACKGROUND: Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. METHODOLOGY/PRINCIPAL FINDINGS: The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name "island integration determinant" (iid). CONCLUSION/SIGNIFICANCE: These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.

Phylogenetic analysis and siderotyping as useful tools in the taxonomy of Pseudomonas stutzeri: description of a novel genomovar.

An examination of the results of phylogenetic analyses based on the sequences of fragments of the 16S rRNA, gyrB and rpoD genes, and the discrimination of genomovars based on siderophore diversity within the genus Pseudomonas, has added important taxonomic tools in the characterization of Pseudomonas stutzeri. Eighteen reference strains, nine newly identified hydrocarbon-degrading strains and three strains showing relevant physiological characteristics of P. stutzeri, together with the type strains of four related species, were included in the study. A novel genomovar within the species is described. A summary of the methodology used in these studies and the results of our attempts to define a solid internal subdivision of this important species within the genus Pseudomonas are presented and discussed.

Siderotyping of fluorescent Pseudomonas: molecular mass determination by mass spectrometry as a powerful pyoverdine siderotyping method.

The numerous pyoverdines so far characterized as siderophores of fluorescent Pseudomonas could be usually differentiated one from each others by the two physico-chemical and physiological methods of siderotyping, i.e., siderophore-isoelectrofocusing and siderophore-mediated iron uptake. As shown in the present paper, the structural diversity of the peptide chain characterizing these molecules results in a very large panel of molecular masses representing 64 different values ranging from 889 to 1,764 Da for the 68 compounds included in the study, with only a few structurally different compounds presenting an identical molecular mass. Thus, the molecular mass determination of pyoverdines through mass spectrometry could be used as a powerful siderotyping method.

Taxonomic heterogeneity, as shown by siderotyping, of strains primarily identified as Pseudomonas putida.

One hundred and forty-four fluorescent pseudomonad strains isolated from various environments (soil, water, plant rhizosphere, hospital) and received as Pseudomonas putida (83 strains), P. putida biovar A (49 strains), P. putida biovar B (10 strains) and P. putida biovar C (2 strains), were analysed by the pyoverdine-isoelectrofocusing and pyoverdine-mediated iron uptake methods of siderotyping. Both methods demonstrated a great diversity among these strains, which could be subdivided into 35 siderovars. Some siderovars specifically included strains that have subsequently been transferred to well-defined Pseudomonas species, e.g. Pseudomonas monteilii or Pseudomonas mosselii, or which could be related by their siderotype to Pseudomonas jessenii or Pseudomonas mandelii. Other siderovars included strains sharing a high level of DNA-DNA relatedness (>70%), thus demonstrating that siderotyping could easily circumscribe strains at the species level. However, a group of seven strains, including the type strain, P. putida ATCC 12633T, were allocated into four siderovars, despite sharing DNA-DNA relatedness values of higher than 70 %. Interestingly, the strong genomic relationships between these seven strains were supported by the structural relationships among their pyoverdines, thus reflecting their phylogenetic affinities. These results strongly support the view that pyoverdine-based siderotyping could be used as a powerful tool in Pseudomonas taxonomy.