A new dimethylsulfoniopropionate lyase of the cupin superfamily in marine bacteria

Dimethylsulfoniopropionate (DMSP) is a marine organosulfur compound with important roles in stress protection, marine biogeochemical cycling, chemical signalling and atmospheric chemistry. Diverse marine microorganisms catabolize DMSP via DMSP lyases to generate the climate-cooling gas and info-chemical dimethyl sulphide. Abundant marine heterotrophs of the Roseobacter group (MRG) are well known for their ability to catabolize DMSP via diverse DMSP lyases. Here, a new DMSP lyase DddU within the MRG strain Amylibacter cionae H-12 and other related bacteria was identified. DddU is a cupin superfamily DMSP lyase like DddL, DddQ, DddW, DddK and DddY, but shares <15% amino acid sequence identity with these enzymes. Moreover, DddU proteins forms a distinct clade from these other cupin-containing DMSP lyases. Structural prediction and mutational analyses suggested that a conserved tyrosine residue is the key catalytic amino acid residue in DddU. Bioinformatic analysis indicated that the dddU gene, mainly from Alphaproteobacteria, is widely distributed in the Atlantic, Pacific, Indian and polar oceans. For reference, dddU is less abundant than dddP, dddQ and dddK, but much more frequent than dddW, dddY and dddL in marine environments. This study broadens our knowledge on the diversity of DMSP lyases, and enhances our understanding of marine DMSP biotransformation.     

Molecular genetic analysis suggesting interactions between AppA and PpsR in regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

The AppA protein plays an essential regulatory role in development of the photosynthetic apparatus in the anoxygenic phototrophic bacterium Rhodobacter sphaeroides 2.4.1 (M. Gomelsky and S. Kaplan, J. Bacteriol. 177:4609-4618, 1995). To gain additional insight into both the role and site of action of AppA in the regulatory network governing photosynthesis gene expression, we investigated the relationships between AppA and other known regulators of photosynthesis gene expression. We determined that AppA is dispensable for development of the photosynthetic apparatus in a ppsR null background, where PpsR is an aerobic repressor of genes involved in photopigment biosynthesis and puc operon expression. Moreover, all suppressors of an appA null mutation thus far isolated, showing improved photosynthetic growth, were found to contain mutations in the ppsR gene. Because ppsR gene expression in R. sphaeroides 2.4.1 appears to be largely independent of growth conditions, we suggest that regulation of repressor activity occurs predominately at the protein level. We have also found that PpsR functions as a repressor not only under aerobic but under anaerobic photosynthetic conditions and thereby is involved in regulating the abundance of the light harvesting complex II, depending on light intensity. It seems likely therefore, that PpsR responds to an integral signal (e.g., changes in redox potential) produced either by changes in oxygen tension or light intensity. The profile of the isolated suppressor mutations in PpsR is in accord with this proposition. We propose that AppA may be involved in a redox-dependent modulation of PpsR repressor activity.     

Isolation and genetic complementation of a sulfolipid-deficient mutant of Rhodobacter sphaeroides.

All photosynthetic organisms are thought to contain the sulfolipid 6-sulfo-alpha-D-quinovosyl diacylglycerol. However, the pathway of sulfolipid biosynthesis has not been elucidated, and the functional or structural significance of this lipid is not known. Mutants of Rhodobacter sphaeroides deficient in sulfolipid accumulation were isolated by directly screening for altered sulfolipid content. The mutants had no apparent phenotype except for the sulfolipid deficiency. A gene, designated sqdA, which complemented one of the mutations was isolated and characterized. The putative sqdA gene product is a protein with a molecular mass of 33.6 kDa that has no sequence similarity to any enzyme of known function.     

Structural and genetic analysis of a mutant of Rhodobacter sphaeroides WS8 deficient in hook length control.

Motility in the photosynthetic bacterium Rhodobacter sphaeroides is achieved by the unidirectional rotation of a single subpolar flagellum. In this study, transposon mutagenesis was used to obtain nonmotile flagellar mutants from this bacterium. We report here the isolation and characterization of a mutant that shows a polyhook phenotype. Morphological characterization of the mutant was done by electron microscopy. Polyhooks were obtained by shearing and were used to purify the hook protein monomer (FlgE). The apparent molecular mass of the hook protein was 50 kDa. N-terminal amino acid sequencing and comparisons with the hook proteins of other flagellated bacteria indicated that the Rhodobacter hook protein has consensus sequences common to axial flagellar components. A 25-kb fragment from an R. sphaeroides WS8 cosmid library restored wild-type flagellation and motility to the mutant. Using DNA adjacent to the inserted transposon as a probe, we identified a 4.6-kb SalI restriction fragment that contained the gene responsible for the polyhook phenotype. Nucleotide sequence analysis of this region revealed an open reading frame with a deduced amino acid sequence that was 23.4% identical to that of FliK of Salmonella typhimurium, the polypeptide responsible for hook length control in that enteric bacterium. The relevance of a gene homologous to fliK in the uniflagellated bacterium R. sphaeroides is discussed.     

Molecular diversity of bacterial production of the climate-changing gas, dimethyl sulphide, a molecule that impinges on local and global symbioses

This paper describes the ddd genes that are involved in theproduction of the gas dimethyl sulphide from the substrate dimethylsulphoniopropionate(DMSP), an abundant molecule that is a stress protectant inmany marine algae and a few genera of angiosperms. What is knownof the arrangement of the ddd genes in different bacteria thatcan undertake this reaction is reviewed here, stressing thefact that these genes are probably subject to horizontal genetransfer and that the same functions (e.g. DMSP transport) maybe accomplished by very different mechanisms. A surprising numberof DMS-emitting bacteria are associated with the roots of higherplants, these including strains of Rhizobium and some rhizospherebacteria in the genus Burkholderia. One newly identified strainthat is predicted to make DMS is B. phymatum which is a highlyunusual β-proteobacterium that forms N₂-fixing noduleson some tropical legumes, in this case, the tree Machaeriumlunatum, which inhabits mangroves. The importance of DMSP catabolismand DMS production is discussed, not only in terms of nutritionalacquisition by the bacteria but also in a speculative scheme(the ‘messy eater’ model) in which the bacteriamay make DMS as an info-chemical to attract other organisms,including invertebrates and other plankton. Key words: Acyl CoA transferase, Burkholderia, CLAW hypothesis, dimethyl sulphide, dimethylsulphoniopropionate, Marinomonas, nitrogen fixation, Rhizobium, rhizosphere, root nodules, Spartina Received 30 May 2007; Revised 27 September 2007 Accepted 1 October 2007     

Phenotypic characterisation and genetic complementation of dimethylsulfoxide respiratory mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract Two chlorate resistant mutants of Rhodobacter sphaeroides were isolated which were deficient in dimethylsulfoxide reductase activity. Immunoblotting experiments showed that the phenotype of these mutants and that of Rhodobacter capsulatus strain DK9, a mutant unable to reduce dimethylsulfoxide, was correlated with low or undetectable levels of the dimethylsulfoxide reductase apoprotein. All three mutants were complemented by a cosmid from a library of Rhodobacter sphaeroides genomic DNA. Further genetic complementation analysis revealed that functions required for restoration of dimethylsulfoxide reductase activity in the Rhodobacter sphaeroides mutants were encoded on an 9 kb EcoR1 DNA fragment derived from this cosmid. Expression of this 9 kb DNA fragment in Escherichia coli showed that it encoded the dimethylsulfoxide reductase structural gene of Rhodobacter sphaeroides .     

Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides.

It has recently become evident that many bacterial respiratory oxidases are members of a superfamily that is related to the eukaryotic cytochrome c oxidase. These oxidases catalyze the reduction of oxygen to water at a heme-copper binuclear center. Fourier transform infrared (FTIR) spectroscopy has been used to examine the heme-copper-containing respiratory oxidases of Rhodobacter sphaeroides Ga. This technique monitors the stretching frequency of CO bound at the oxygen binding site and can be used to characterize the oxidases in situ with membrane preparations. Oxidases that have a heme-copper binuclear center are recognizable by FTIR spectroscopy because the bound CO moves from the heme iron to the nearby copper upon photolysis at low temperature, where it exhibits a diagnostic spectrum. The FTIR spectra indicate that the binuclear center of the R. sphaeroides aa3-type cytochrome c oxidase is remarkably similar to that of the bovine mitochondrial oxidase. Upon deletion of the ctaD gene, encoding subunit I of the aa3-type oxidase, substantial cytochrome c oxidase remains in the membranes of aerobically grown R. sphaeroides. This correlates with a second wild-type R. sphaeroides is grown photosynthetically, the chromatophore membranes lack the aa3-type oxidase but have this second heme-copper oxidase. Subunit I of the heme-copper oxidase superfamily contains the binuclear center. Amino acid sequence alignments show that this subunit is structurally very highly conserved among both eukaryotic and prokaryotic species. The polymerase chain reaction was used to show that the chromosome of R. sphaeroides contains at least one other gene that is a homolog of ctaD, the gene encoding subunit I of the aa3-type cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular architecture of photosynthetic membranes in Rhodobacter sphaeroides: the role of PufX

The effects of the PufX polypeptide on membrane architecture were investigated by comparing the composition and structures of photosynthetic membranes from PufX+ and PufX- strains of Rhodobacter sphaeroides. We show that this single polypeptide profoundly affects membrane morphology, leading to highly elongated cells containing extended tubular membranes. Purified tubular membranes contain helical arrays composed solely of dimeric RC-LH1-PufX (RC, reaction centre; LH, light harvesting) complexes with apparently open LH1 rings. PufX- cells contain crystalline membranes with a pseudo-hexagonal packing of monomeric core complexes. Analysis of purified complexes by electron microscopy and atomic force microscopy shows that LH1 and PufX form a continuous ring of protein around each RC. A model of the tubular membrane is presented with PufX located adjacent to the stained region created by a vacant LH1beta. This arrangement, coupled with a flexible ring, would give the RC QB site transient access to the interstices in the lattice, which might be of functional importance. We discuss the implications of our data for the export of quinol from the RC, for eventual reduction of the cytochrome bc1 complex.     

Quantitative proteomic analysis of intracytoplasmic membrane development in Rhodobacter sphaeroides

The purple phototrophic bacteria elaborate a specialized intracytoplasmic membrane (ICM) system for the conversion of solar energy to ATP. Previous radiolabelling and ultrastructural experiments have shown that ICM assembly in Rhodobacter sphaeroides is initiated at indentations of the cytoplasmic membrane, termed UPB. Here, we report proteomic analyses of precursor (UPB) and mature (ICM) fractions. Qualitative data identified 387 proteins, only 43 of which were found in the ICM, reflecting its specialized role within the cell, the conversion of light into chemical energy; 236 proteins were found in the significantly more complex UPB proteome. Metabolic labelling was used to quantify the relative distribution of 173 proteins between the UPB and ICM fractions. Quantification reveals new information on assembly of the RC-LH1-PufX, ATP synthase and NAD(P)H transhydrogenase complexes, as well as showing that the UPB is enriched in enzymes for lipid, carbohydrate and amino acid metabolism, tetrapyrrole biosynthesis and proteins representing a wide range of other metabolic and biosynthetic functions. Proteins involved in light harvesting, photochemistry, electron transport and ATP synthesis are all enriched in ICM, consistent with the spatial proximity of energy capturing and transducing functions. These data provide further support to the developmental precursor-product relationship between UPB and ICM.     

Identification and molecular genetic analysis of multiple loci contributing to high-level tellurite resistance in Rhodobacter sphaeroides 2.4.1.

The ability of the facultative photoheterotroph Rhodobacter sphaeroides to tolerate and reduce high levels of tellurite in addition to at least 10 other rare earth metal oxides and oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. We report the identification and characterization of two loci involved in high-level tellurite resistance. The first locus contains four genes, two of which, trgAB, confer increased tellurite resistance when introduced into the related bacterium Paracoccus denitrificans. The trgAB-derived products display no significant homology to known proteins, but both are likely to be membrane-associated proteins. Immediately downstream of trgB, the cysK (cysteine synthase) and orf323 genes were identified. Disruption of the cysK gene resulted in decreased tellurite resistance in R. sphaeroides, confirming earlier observations on the importance of cysteine metabolism for high-level tellurite resistance. The second locus identified is represented by the telA gene, which is separated from trgAB by 115 kb. The telA gene product is 65% similar to the product of the klaB (telA) gene from the tellurite-resistance-encoding kilA operon from plasmid RK2. The genes immediately linked to the R. sphaeroides telA gene have no similarity to other components of the kilA operon. R. sphaeroides telA could not functionally substitute for the plasmid RK2 telA gene, indicating substantial functional divergence between the two gene products. However, inactivation of R. sphaeroides telA resulted in a significant decrease in tellurite resistance compared to the wild-type strain. Both cysK and telA null mutations readily gave rise to suppressors, suggesting that the phenomenon of high-level tellurite resistance in R. sphaeroides is complex and other, as yet uncharacterized, loci may be involved.     

Molecular cloning of the 5-aminolevulinic acid dehydratase gene from Rhodobacter sphaeroides.

A hemB mutant of Escherichia coli was used to clone the gene encoding 5-aminolevulinic acid dehydratase from Rhodobacter sphaeroides after physiological complementation of the mutation. A 2.9-kb DNA fragment was obtained and cloned in both orientations into the unique PstI restriction site of pUC19. This recombinant plasmid encodes a protein (Mr 39,000) that is immunoreactive with antibodies raised against the enzyme from higher plants.     

Identification and characterization of a GroEL homologue in Rhodobacter sphaeroides

A protein closely related to the Escherichia coli GroEL protein has been isolated from Rhodobacter sphaeroides. Native and SDS-polyacrylamide gel electrophoresis of this protein have shown that it is present in the cell as a multimeric complex of Mr 670,000 which is composed of a monomer of Mr 58,000. Antisera raised against the Mr 58,000 polypeptide have been shown to cross-react with GroEL and the alpha subunit of the pea plastid chaperonin. The N-terminal amino acid sequence of the Mr 58,000 polypeptide is identical to that of GroEL at 15 of 19 residues and is also closely related to the alpha subunit of the pea plastid chaperonin, though less so to the beta subunit.