Mineralization of small saline and soda lakes can vary significantly depending on the season and weather conditions. The ability to rapidly adjust intracellular concentrations of key osmolytes (also known as compatible solutes) to changes in external salinity is an important property of microorganisms inhabiting these biotopes (8,18,34). Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) was found to be a major compatible solute in many halophilic or halotolerant bacteria isolated from alkaline, moderately hypersaline environments (14). This organic solute can be synthesized de novo or taken up from the environment when available (15, 18). The biochemistry and genetics of ectoine synthesis have been described for several bacteria (15,28,29,32). However, little is known about the transcriptional regulation of the ectoine biosynthetic pathway. Comprehensive analysis of the ectoine gene cluster ectABC in Chromohalobacter salexigens showed four putative transcription initiation sites upstream of the ectA start codon. Two 70 -dependent, one S -dependent, and one 32 -dependent promoter were identified and shown to be involved in ectABC transcription in this bacterium (6). Transcription of the ectA, ectB, and ectC genes from Marinococcus halophilus was initiated from three individual 70 / A -dependent promoter sequences located upstream of each gene (3). In Bacillus pasteurii, the ectABC genes are organized in a single operon preceded by a typical A -dependent promoter region (21). The halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z is capable of growth at a salinity as high as 2 M NaCl (19). It was demonstrated that in response to the elevated salinity of the growth medium, M. alcaliphilum cells accumulate ectoine as a major osmoprotective compound (20). The ectoine biosynthesis pathway in M. alcaliphilum 20Z is similar to the pathway employed by halophilic/halotolerant heterotrophs and involves three specific enzymes: diaminobutyric acid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA), and ectoine synthase (EctC) (7,21,24,30,32,49). In M. alcaliphilum 20Z, the ectoine biosynthetic genes were shown to be organized in the ectABC-ask operon containing the additional ask gene, encoding aspartokinase (32). Here we describe the transcriptional organization of the ectoine biosynthetic genes in M. alcaliphilum 20Z. We identify a new MarR-like transcriptional regulator (EctR1) and show that EctR1 represses the expression of the ectABC-ask operon from the ectAp 1 promoter by binding at the putative Ϫ10 sequence. These results demonstrate the presence of a new, previously uncharacterized regulatory system for ectoine biosynthesis in the salt-tolerant methanotroph. MATERIALS AND METHODSBacterial strains and culture conditions. The M. alcaliphilum and Escherichia coli strains, plasmids, and primers used in this study are listed in Tables S1 and S2 in the supplemental material. M. alcaliphilum strains were grown at 30°C under a methane-air atmosphere (1:1) or in the presence of 0....

We investigated phenotypes of mutants of Methylotenera mobilis JLW8 with lesions in genes predicted to encode functions of the denitrification pathway, as well as mutants with mutations in methanol dehydrogenase-like structural genes xoxF1 and xoxF2, in order to obtain insights into denitrification and methanol metabolism by this bacterium. By monitoring the accumulation of nitrous oxide, we demonstrate that a periplasmic nitrate reductase, NAD(P)-linked and copper-containing nitrite reductases, and a nitric oxide reductase are involved in the denitrification pathway and that the pathway must be operational in aerobic conditions. However, only the assimilatory branch of the denitrification pathway was essential for growth on methanol in nitrate-supplemented medium. Mutants with mutations in each of the two xoxF genes maintained their ability to grow on methanol, but not the double XoxF mutant, suggesting that XoxF proteins act as methanol dehydrogenase enzymes in M. mobilis JLW8. Reduced levels of nitrous oxide accumulated by the XoxF mutants compared to the wild type suggest that these enzymes must be capable of donating electrons for denitrification.

No abstract

An active pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) from the thermotolerant methanotroph Methylococcus capsulatus Bath, containing a six-residue polyhistidine tag, was characterized. The enzyme was homodimeric (2 x 45 kDa), nonallosteric and most active at pH 7.0. PPi-PFK catalyzed reactions of PPi-dependent phosphorylation of fructose-6-phosphate (F-6-P) (K(m) 2.27 mM and V(max) 7.6 U mg(-1) of protein), sedoheptulose-7-phosphate (K(m) 0.027 mM and V(max) 31 U mg(-1)) and ribulose-5-phosphate. In the reaction with F-6-P, the apparent K(m) for PPi was 0.027 mM, while in the reverse reaction, K(m) for orthophosphate was 8.69 mM and that for fructose-1,6-bisphosphate 0.328 mM (V(max) 9.0 U mg(-1)). Phylogenetically, M. capsulatus PPi-PFK was most similar to PPi-PFKs from the lithoautotrophic ammonia oxidizers Nitrosomonas europaea (74.0%), Nitrosospira multiformis (73.6%) and Betaproteobacterial methylotroph Methylibium petroleiphilum PM1 (71.6% identity). Genes coding PPi-PFK and a putative V-type H(+)-translocating pyrophosphatase (H(+)-PPi-ase) were cotranscribed as an operon. The potential significance of the PPi-PFK for regulation of carbon and energy fluxes in M. capsulatus Bath is discussed.

The genes of ectoine biosynthesis pathway were identified in six species of aerobic, slightly halophilic bacteria utilizing methane, methanol or methylamine. Two types of ectoine gene cluster organization were revealed in the methylotrophs. The gene cluster ectABC coding for diaminobutyric acid (DABA) acetyltransferase (EctA), DABA aminotransferase (EctB) and ectoine synthase (EctC) was found in methanotrophs Methylobacter marinus 7C and Methylomicrobium kenyense AMO1(T). In methanotroph Methylomicrobium alcaliphilum ML1, methanol-utilizers Methylophaga thalassica 33146(T) , Methylophaga alcalica M8 and methylamine-utilizer Methylarcula marina h1(T), the genes forming the ectABC-ask operon are preceded by ectR, encoding a putative transcriptional regulatory protein EctR. Phylogenetic relationships of the Ect proteins do not correlate with phylogenetic affiliation of the strains, thus implying that the ability of methylotrophs to produce ectoine is most likely the result of a horizontal transfer event.

The review summarizes the data on the metabolic potential of methanotrophs as producers of biopolymers, alternative biofuel, bioprotectants, and other secondary metabolites. The work provides the examples of modern 'omic' technologies used for genetic engineering of efficient methanotrophic producers.

Aerobic bacteria utilizing methane as the carbon and energy source do not use sugars as growth substrates but possess the gene coding for glucokinase (Glk), an enzyme converting glucose into glucose 6-phosphate. Here we demonstrate the functionality and properties of Glk from an obligate methanotroph Methylomicrobium alcaliphilum 20Z. The recombinant Glk obtained by heterologous expression in Escherichia coli was found to be close in biochemical properties to other prokaryotic Glks. The homodimeric enzyme (2 × 35 kDa) catalyzed ATP-dependent phosphorylation of glucose and glucosamine with nearly equal activity, being inhibited by ADP (K = 2.34 mM) but not affected by glucose 6-phosphate. Chromosomal deletion of the glk gene resulted in a loss of Glk activity and retardation of growth as well as in a decrease of intracellular glycogen content. Inactivation of the genes encoding sucrose phosphate synthase or amylosucrase, the enzymes involved in glycogen biosynthesis via sucrose as intermediate, did not prevent glycogen accumulation. In silico analysis revealed glk orthologs predominantly in methanotrophs harboring glycogen synthase genes. The data obtained suggested that Glk is implicated in the regulation of glycogen biosynthesis/degradation in an obligate methanotroph.

No abstract