Transposon mutagenesis for methylotrophic bacteria using Methylorubrum extorquens AM1 as a model system.

Transposon mutagenesis utilizes transposable genetic elements that integrate into a recipient genome to generate random insertion mutations which are easily identified. This forward genetic approach has proven powerful in elucidating complex processes, such as various pathways in methylotrophy. In the past decade, many methylotrophic bacteria have been shown to possess alcohol dehydrogenase enzymes that use lanthanides (Lns) as cofactors. Using Methylorubrum extorquens AM1 as a model organism, we discuss the experimental designs, protocols, and results of three transposon mutagenesis studies to identify genes involved in different aspects of Ln-dependent methanol oxidation. These studies include a selection for transposon insertions that prevent toxic intracellular formaldehyde accumulation, a fluorescence-imaging screen to identify regulatory processes for a primary Ln-dependent methanol dehydrogenase, and a phenotypic screen for genes necessary for function of a Ln-dependent ethanol dehydrogenase. We anticipate that the methods described in this chapter can be applied to understand other metabolic systems in diverse bacteria.

Gene products and processes contributing to lanthanide homeostasis and methanol metabolism in Methylorubrum extorquens AM1.

Lanthanide elements have been recently recognized as "new life metals" yet much remains unknown regarding lanthanide acquisition and homeostasis. In Methylorubrum extorquens AM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate. This property enabled a transposon mutagenesis study and growth studies to confirm novel gene products required for XoxF1 function. The identified genes encode an MxaD homolog, an ABC-type transporter, an aminopeptidase, a putative homospermidine synthase, and two genes of unknown function annotated as orf6 and orf7. Lanthanide transport and trafficking genes were also identified. Growth and lanthanide uptake were measured using strains lacking individual lanthanide transport cluster genes, and transmission electron microscopy was used to visualize lanthanide localization. We corroborated previous reports that a TonB-ABC transport system is required for lanthanide incorporation to the cytoplasm. However, cells were able to acclimate over time and bypass the requirement for the TonB outer membrane transporter to allow expression of xoxF1 and growth. Transcriptional reporter fusions show that excess lanthanides repress the gene encoding the TonB-receptor. Using growth studies along with energy dispersive X-ray spectroscopy and transmission electron microscopy, we demonstrate that lanthanides are stored as cytoplasmic inclusions that resemble polyphosphate granules.

Draft Genome Sequence of the Caffeine-Degrading Methylotroph Methylorubrum populi Pinkel.

A pink-pigmented facultative methylotroph, Methylorubrum populi Pinkel, was isolated from compost by selective enrichment with caffeine (3,5,7-trimethylxanthine) as the sole carbon, nitrogen, and energy source. We report here its high-quality draft genome sequence, assembled in 35 contigs totaling 5,630,907 bp. We identified 5,681 protein-coding sequences, including those putatively involved in caffeine degradation.

Chemotaxis of Pseudomonas putida F1 to Alcohols Is Mediated by the Carboxylic Acid Receptor McfP.

Although alcohols are toxic to many microorganisms, they are good carbon and energy sources for some bacteria, including many pseudomonads. However, most studies that have examined chemosensory responses to alcohols have reported that alcohols are sensed as repellents, which is consistent with their toxic properties. In this study, we examined the chemotaxis of Pseudomonas putida strain F1 to n-alcohols with chain lengths of 1 to 12 carbons. P. putida F1 was attracted to all n-alcohols that served as growth substrates (C2 to C12) for the strain, and the responses were induced when cells were grown in the presence of alcohols. By assaying mutant strains lacking single or multiple methyl-accepting chemotaxis proteins, the receptor mediating the response to C2 to C12 alcohols was identified as McfP, the ortholog of the P. putida strain KT2440 receptor for C2 and C3 carboxylic acids. Besides being a requirement for the response to n-alcohols, McfP was required for the response of P. putida F1 to pyruvate, l-lactate, acetate, and propionate, which are detected by the KT2440 receptor, and the medium- and long-chain carboxylic acids hexanoic acid and dodecanoic acid. ß-Galactosidase assays of P. putida F1 carrying an mcfP-lacZ transcriptional fusion showed that the mcfP gene is not induced in response to alcohols. Together, our results are consistent with the idea that the carboxylic acids generated from the oxidation of alcohols are the actual attractants sensed by McfP in P. putida F1, rather than the alcohols themselves.IMPORTANCE Alcohols, released as fermentation products and produced as intermediates in the catabolism of many organic compounds, including hydrocarbons and fatty acids, are common components of the microbial food web in soil and sediments. Although they serve as good carbon and energy sources for many soil bacteria, alcohols have primarily been reported to be repellents rather than attractants for motile bacteria. Little is known about how alcohols are sensed by microbes in the environment. We report here that catabolizable n-alcohols with linear chains of up to 12 carbons serve as attractants for the soil bacterium Pseudomonas putida, and rather than being detected directly, alcohols appear to be catabolized to acetate, which is then sensed by a specific cell-surface chemoreceptor protein.

Pyrroloquinoline Quinone Ethanol Dehydrogenase in Methylobacterium extorquens AM1 Extends Lanthanide-Dependent Metabolism to Multicarbon Substrates.

Lanthanides are utilized by microbial methanol dehydrogenases, and it has been proposed that lanthanides may be important for other type I alcohol dehydrogenases. A triple mutant strain (mxaF xoxF1 xoxF2; named MDH-3), deficient in the three known methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF, was mutated in the MDH-3 background, the quadruple mutant strain could no longer grow on methanol in minimal medium with added lanthanum (La3+). ExaF was purified from cells grown with both calcium (Ca2+) and La3+ and with Ca2+ only, and the protein species were studied biochemically. Purified ExaF is a 126-kDa homodimer that preferentially binds La3+ over Ca2+ in the active site. UV-visible spectroscopy indicates the presence of pyrroloquinoline quinone (PQQ) as a cofactor. ExaF purified from the Ca2+-plus-La3+ condition readily oxidizes ethanol and has secondary activities with formaldehyde, acetaldehyde, and methanol, whereas ExaF purified from the Ca2+-only condition has minimal activity with ethanol as the substrate and activity with methanol is not detectable. The exaF mutant is not affected for growth with ethanol; however, kinetic and in vivo data show that ExaF contributes to ethanol metabolism when La3+ is present, expanding the role of lanthanides to multicarbon metabolism. IMPORTANCE: ExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxF-type alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases.

Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth.

UNLABELLED: Methylobacterium extorquens AM1 has two distinct types of methanol dehydrogenase (MeDH) enzymes that catalyze the oxidation of methanol to formaldehyde. MxaFI-MeDH requires pyrroloquinoline quinone (PQQ) and Ca in its active site, while XoxF-MeDH requires PQQ and lanthanides, such as Ce and La. Using MeDH mutant strains to conduct growth analysis and MeDH activity assays, we demonstrate that M. extorquens AM1 has at least one additional lanthanide-dependent methanol oxidation system contributing to methanol growth. Additionally, the abilities of different lanthanides to support growth were tested and strongly suggest that both XoxF and the unknown methanol oxidation system are able to use La, Ce, Pr, Nd, and, to some extent, Sm. Further, growth analysis using increasing La concentrations showed that maximum growth rate and yield were achieved at and above 1 µM La, while concentrations as low as 2.5 nM allowed growth at a reduced rate. Contrary to published data, we show that addition of exogenous lanthanides results in differential expression from the xox1 and mxa promoters, upregulating genes in the xox1 operon and repressing genes in the mxa operon. Using transcriptional reporter fusions, intermediate expression from both the mxa and xox1 promoters was detected when 50 to 100 nM La was added to the growth medium, suggesting that a condition may exist under which M. extorquens AM1 is able to utilize both enzymes simultaneously. Together, these results suggest that M. extorquens AM1 actively senses and responds to lanthanide availability, preferentially utilizing the lanthanide-dependent MeDHs when possible. IMPORTANCE: The biological role of lanthanides is a nascent field of study with tremendous potential to impact many areas in biology. Our studies demonstrate that there is at least one additional lanthanide-dependent methanol oxidation system, distinct from the MxaFI and XoxF MeDHs, that may aid in classifying additional environmental organisms as methylotrophs. Further, our data suggest that M. extorquens AM1 has a mechanism to regulate which MeDH is transcribed, depending on the presence or absence of lanthanides. While the mechanism controlling differential regulation is not yet understood, further research into how methylotrophs obtain and use lanthanides will facilitate their cultivation in the laboratory and their use as a biomining and biorecycling strategy for recovery of these commercially valuable rare-earth elements.