Taxing debate for taxonomists.
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Biomedical subjects
Publications and source records attributed to H Gest.
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On September 7, 1999, the 200th anniversary of the death of Dr Jan Ingen-Housz was commemorated by ceremonies in Calne, England. Ingen-Housz discovered the action of light in photosynthesis in 1779, following Joseph Priestley's demonstration that green plants had the capacity to produce oxygen. Priestley's claim for priority in discovering the light requirement of photosynthesis is examined.
The purple photosynthetic bacterium Rhodospirillum centenum is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation. To identify components specific for photoperception, we conducted mini-Tn5-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity. A majority of the ca. 250 mutants identified lost the ability to form motile swarm cells on an agar surface. These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella. Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus. Several photosensory mutants also were obtained with defects in the perception and transmission of light signals. Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception. It was shown that screening for photosensory defective R. centenum swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria.
The chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum contains five open reading frames (ORFs) that have significant sequence homology to chemotaxis genes from other bacteria. To elucidate the functions of each ORF, we have made various mutations in the gene cluster and analyzed their phenotypic defects. Deletion of the entire che operon (delta che), as well as nonpolar disruptions of cheAY, cheW, and cheR, resulted in a smooth-swimming phenotype, whereas disruption of cheB resulted in a locked tumbly phenotype. Each of these mutants was defective in chemotactic response. Interestingly, disruption of cheY resulted in a slight increase in the frequency of tumbling/reversal with no obvious defects in chemotactic response. In contrast to observations with Escherichia coli and several other bacteria, we found that all of the che mutant cells were capable of differentiating into hyperflagellated swarmer cells when plated on a solid agar surface. When viewed microscopically, the smooth-swimming che mutants exhibited active surface motility but were unable to respond to a step-down in light intensity. Both positive and negative phototactic responses were abolished in all che mutants, including the cheY mutant. These results indicate that eubacterial photosensory perception is mediated by light-generated signals that are transmitted through the chemotaxis signal transduction cascade.
Most photosynthetic microorganisms have the capability of photosensing light quality and intensity. Movement of motile photosynthetic microorganisms to locales that offer optimal physical and chemical conditions for light-dependent growth provides obvious selective advantages. Among phototrophs, many cyanobacteria and algae migrate towards or away from the direction of light, a process termed phototaxis. In contrast, anoxygenic photosynthetic bacteria are believed to respond to changes in light intensity in a manner that is not related to the direction of light, a process that is often described by the term "photophobic". We recently reported that "swarm colonies" of the purple photosynthetic bacterium Rhodospirillum centenum are capable of macroscopically visible phototactic behavior. In the present study we further characterize the phototactic behavior of R. centenum swarm colonies and provide an action spectrum that delineates regions of the spectrum that elicit positive and negative phototaxis.
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The original bidirectional compound chemostat (gradostat) described by Lovitt and Wimpenny has been simplified by making a more compact apparatus in which chemical gradients are established by diffusion between adjacent culture chambers. The experimental model (diffusion coupled (DC) gradostat) consisted of five chambers whose contents could be agitated by turbines rotating in the horizontal plane on a common shaft. Two biological experiments were designed to reveal the value of the DC gradostat. A methylotroph (Methylophilus methylotrophus) grown in a methanol gradient showed expected changes in cell viability as a function of position in the five vessel array. Cells of two species of photosynthetic bacteria (Rhodobacter capsulata and Rhodopseudomonas marina/agilis) with different salt sensitivities could be mixed and subsequently separated by the DC gradostat operating with a NaCl gradient of 0-3% w/v.
Intraspecies and intergenus complementation analysis were utilized to demonstrate that photosynthesis genes are clustered in distantly related purple photosynthetic bacteria. Specifically, we show that the linkage order for genes involved in bacteriochlorophyll and carotenoid biosynthesis in Rhodospirillum centenum are arranged essentially as in Rhodobacter capsulatus and Rhodobacter sphaeroides. In addition, the location and relative distance observed between the puf and puh operons which encode for light harvesting and reaction-centre structural genes are also conserved between these species. Conservation of the photosynthesis gene cluster implies either that there are structural or regulatory constraints that limit rearrangement of the photosynthesis gene cluster or that there may have been lateral transfer of the photosynthesis gene cluster among different species of phototrophic bacteria.
A genetic system has been developed for studying bacterial photosynthesis in the recently described nonsulfur purple photosynthetic bacterium Rhodospirillum centenum. Nonphotosynthetic mutants of R. centenum were obtained by enrichment for spontaneous mutations, by ethyl methanesulfonate mutagenesis coupled to penicillin selection on solid medium, and by Tn5 transposition mutagenesis with an IncP plasmid vector containing a temperature-sensitive origin of replication. In vivo and in vitro characterization of individual strains demonstrated that 38 strains contained mutations that blocked bacteriochlorophyll a biosynthesis at defined steps of the biosynthetic pathway. Collectively, these mutations were shown to block seven of eight steps of the pathway leading from protoporphyrin IX to bacteriochlorophyll a. Three mutants were isolated in which carotenoid biosynthesis was blocked early in the biosynthetic pathway; the mutants also exhibited pleiotropic effects on stability or assembly of the photosynthetic apparatus. Five mutants failed to assemble a functional reaction center complex, and seven mutants contained defects in electron transport as shown by an alteration in cytochromes. In addition, several regulatory mutants were isolated that acquired enhanced repression of bacteriochlorophyll in response to the presence of molecular oxygen. The phenotypes of these mutants are discussed in relation to those of similar mutants of Rhodobacter and other Rhodospirillum species of purple photosynthetic bacteria.
Rhodospirillum centenum resembles typical nonsulfur photosynthetic bacteria in a number of respects, including its ability to grow either anaerobically as a phototroph or aerobically as a heterotroph. We demonstrate, however, that R. centenum is unusual in its ability to synthesize a functional photosynthetic apparatus regardless of the presence of molecular oxygen. Aerobically expressed photopigments were shown to be functionally active, as demonstrated by the ability of heterotrophically grown cells to grow photosynthetically, without a lag, when suddenly placed under anaerobic conditions. An R. centenum mutant that has acquired the ability to repress synthesis of photopigments in the presence of oxygen was also characterized. Both the wild type and the oxygen-repressed mutant of R. centenum were found to exhibit high light intensity repression of photopigment biosynthesis. The latter result suggests that R. centenum contains separate regulatory circuits for controlling synthesis of its photochemical apparatus by light intensity and oxygen.
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A novel non-sulfur purple photosynthetic bacterium, designated Rhodospirillum centenum, was isolated from an enrichment culture designed to favor growth of anoxygenic photosynthetic N2-fixing bacteria. R. centenum grows optimally at 40-42 degrees C and has the capacity to produce cytoplasmic 'R bodies', refractile structures not observed hitherto in photosynthetic prokaryotes. The bacterium is also unusual among photosynthetic bacteria in that it forms desiccation-resistant cysts when grown aerobically in darkness with butyrate as the sole carbon source.
Purple phototrophic bacteria of the genus Chromatium can grow as either photoautotrophs or photoheterotrophs. To determine the growth mode of the thermophilic Chromatium species, Chromatium tepidum, under in situ conditions, we have examined the carbon isotope fractionation patterns in laboratory cultures of this organism and in mats of C. tepidum which develop in sulfide thermal springs in Yellowstone National Park. Isotopic analysis (13C/12C) of total carbon, carotenoid pigments, and bacteriochlorophyll from photoautotrophically grown cultures of C. tepidum yielded 13C fractionation factors near -20%. Cells of C. tepidum grown on excess acetate, wherein synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase ribulose bisphosphate carboxylase) was greatly repressed, were isotopically heavier, fractionation factors of ca. -7% being observed. Fractionation factors determined by isotopic analyses of cells and pigment fractions of natural populations of C. tepidum growing in three different sulfide thermal springs in Yellowstone National Park were approximately -20%, indicating that this purple sulfur bacterium grows as a photoautotroph in nature.
Normal sulfur isotope effects averaging epsilon = -5.2 +/- 1.4% (s.d.) were consistently observed for the oxidation of sulfide in aqueous solution. Reaction products were sulfate, thiosulfate and sulfite at pH 10.8-11 in distilled water; S0 was formed in two experiments with synthetic seawater at pH 8-9.5. Because the -5.2% normal isotope effect differs significantly from the previously measured +2% inverse effect associated with anaerobic oxidation of sulfide by photosynthetic bacteria, stable sulfur isotopic measurements are potentially useful for distinguishing aerobic vs. anaerobic sulfide oxidation in marine and freshwater sulfureta.
Stable isotopic distributions in the sulfur cycle were studied with pure and mixed cultures of the anaerobic bacteria, Chlorobium vibrioforme and Desulfovibrio vulgaris. D. vulgaris and C. vibrioforme can catalyze three reactions constituting a complete anaerobic sulfur cycle: reduction of sulfate to sulfide (D. vulgaris), oxidation of sulfide to elemental sulfur (C. vibrioforme), and oxidation of sulfur to sulfate (C. vibrioforme). In all experiments, the first and last reactions favored concentration of the light 32S isotope in products (isotopic fractionation factor epsilon = -7.2 and -1.7%, respectively), whereas oxidation of sulfide favored concentration of the heavy 34S isotope in products (epsilon = +1.7%). Experimental results and model calculations suggest that elemental sulfur enriched in 34S versus sulfide may be a biogeochemical marker for the presence of sulfide-oxidizing bacteria in modern and ancient environments.