Jane Gibson

Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris.

Rhodopseudomonas palustris is among the most metabolically versatile bacteria known. It uses light, inorganic compounds, or organic compounds, for energy. It acquires carbon from many types of green plant–derived compounds or by carbon dioxide fixation, and it fixes nitrogen. Here we describe the genome sequence of R. palustris, which consists of a 5,459,213-base-pair (bp) circular chromosome with 4,836 predicted genes and a plasmid of 8,427 bp. The sequence reveals genes that confer a remarkably large number of options within a given type of metabolism, including three nitrogenases, five benzene ring cleavage pathways and four light harvesting 2 systems. R. palustris encodes 63 signal transduction histidine kinases and 79 response regulator receiver domains. Almost 15% of the genome is devoted to transport. This genome sequence is a starting point to use R. palustris as a model to explore how organisms integrate metabolic modules in response to environmental perturbations.

A phylogenetic analysis of the purple photosynthetic bacteria

Seven species of purple photosynthetic bacteria have been characerized by oligonucleotide cataloging of their 16S ribosomal RNAs. The relationships so revealed among them do not agree well with their classical taxonomic classification. However, they are in agreement with those derived from comparative analysis of cytochromec sequences. Since the two macromolecules, rRNA and cytochromec, are functionally unrelated, the agreement between the two methods virtually rules out lateral gene transfer as the cause of either result. The patterns seen reflect the true genealogies of the organisms. The purple photosynthetic bacteria constitute a major phylogenetic unit—apparently as extensive as the Gram-positive bacteria—but a unit that is not phylogenetically isolated. Intermixed genealogically with these photosynthetic species are many classically recognized nonphotosynthetic Gram-negative organisms. A number of the latter have specific relatives within the photosynthetic cluster.

Pseudomonas aeruginosa-Candida albicans Interactions: Localization and Fungal Toxicity of a Phenazine Derivative

ABSTRACT Phenazines are redox-active small molecules that play significant roles in the interactions between pseudomonads and diverse eukaryotes, including fungi. When Pseudomonas aeruginosa and Candida albicans were cocultured on solid medium, a red pigmentation developed that was dependent on P. aeruginosa phenazine biosynthetic genes. Through a genetic screen in combination with biochemical experiments, it was found that a P. aeruginosa-produced precursor to pyocyanin, proposed to be 5-methyl-phenazinium-1-carboxylate (5MPCA), was necessary for the formation of the red pigmentation. The 5MPCA-derived pigment was found to accumulate exclusively within fungal cells, where it retained the ability to be reversibly oxidized and reduced, and its detection correlated with decreased fungal viability. Pyocyanin was not required for pigment formation or fungal killing. Spectral analyses showed that the partially purified pigment from within the fungus differed from aeruginosins A and B, two red phenazine derivatives formed late in P. aeruginosa cultures. The red pigment isolated from C. albicans that had been cocultured with P. aeruginosa was heterogeneous and difficult to release from fungal cells, suggesting its modification within the fungus. These findings suggest that intracellular targeting of some phenazines may contribute to their toxicity and that this strategy could be useful in developing new antifungals.

Phosphate utilization and alkaline phosphatase activity in Anacystis nidulans (Synechococcus)

Anacystis nidulans (Synechococcus) was maintained in a medium of low phosphate concentration (0.1 mM) and grew with a normal doubling time of 5 hrs at 30°C. Such cultures ahd a normal pigment composition and alkaline phosphatase was detectable at low specific activities only.The onset of phosphate-limited growth occurred when the phosphate concentration in the medium fell to a value below 4 μM (the limit of accurate determination by the assay method used) and resulted in increases in alkaline phosphatase activity, reaching a final 10 to 15 fold increase in specific activity after a period of several hours. Marked changes in the overall pigment composition occurred in this period of growth restriction. The addition of phosphate to such cultures resulted in a halt in synthesis of the enzyme and the restoration of normal pigmentation before growth resumed at the normal rate.Several organic phosphate esters could replace inorganic phosphate for growth and were also hydrolyzed by the partially purified enzyme, but growth rates were characteristically lower and the specific activity only 3 to 4 fold higher than in cultures grown in phosphate excess.Studies with the partially purified enzyme suggested that it differed in some of its properties from other alkaline phosphatases described in the literature.

The Phylogeny of the Green Photosynthetic Bacteria: Absence of a Close Relationship Between Chlorobium and Chloroflexus

Summary Ribosmal RNA oligonucleotide cataloging has been used to explore the phylogenetic relationships among the following green photosynthetic bacteria and their relatives: Chlorobium vibrioforme, C. limicola, Prosthecochloris aestuarii, Chloroherpeton thalassium, Chloroflexus aurantiacus, Herpetosiphon aurantiacus, H. strain Senghas and Thermomicrobium roseum . The first four species form a coherent, moderately tight phylogenetic grouping, the last four a coherent, but looser grouping. No evidence exists for a specific relationship between these two groupings, however. Each can be considered to represent a eubacterial “phylum”.

Ammonia uptake and retention in some cyanobacteria

The internal pool of ammonia in strains of unicellular and filamentous cyanobacteria was found to be 6–12 nmol·mg-1 protein. In nitrate grown Anacystis nidulans R-2 the pool size averaged 12 nmol·mg-1 protein, which corresponds to 2.3 mM, and was little affected by N-source or medium pH during growth. Cells from NH4+-limited continuous culture contained comparable pools, and cell yield was independent of medium pH (7.2–8.5). The internal pool was not bound to macromolecules. The pool fell transiently to about one-third within 2 h on shifting cells to N-free medium, but was slowly regenerated over 24 h.Added ammonia was removed from solution by illuminated cell suspensions at a linear rate, adequate to supply biosynthetic needs, to residual concentrations less than 5 μM. An apparent Km of less than 1 μM can be inferred. Uptake rates were independent of N-source during growth, and of assay pH over the range 6.2–8.7. Bicarbonate was needed for uptake, but the rate of uptake was not influenced by the simultaneous presence of NaNO3 (10 mM) or CH3NH3Cl (0.15 mM). Uptake was energydependent, and was eliminated in dark, anaerobic conditions or by the addition of protonophores. Uptake was also strongly inhibited by dicyclohexylearbodiimide, an ATPase inhibitor, by — SH reagents and methionine sulfoximine, suggesting that interference with energy supply or with ammonia metabolism prevented further entry into the cells.

CO2 fixation and its regulation in Anacystis nidulans (Synechococcus).

Anacystis nidulans (Synechococcus) had a minimal doubling time of 5 hrs at 30°C at saturating light intensity and carbon dioxide concentration. Half maximal growth rates in saturating CO2 occurred at a light intensity of 0.54 mW per cm2, and there was an apparent threshold intensity of 0.13 mW per cm2 below which no growth occurred. Growth rate in saturating light was dependent on the concentration of CO2+H2CO3 in the medium, rather than on total dissolved CO2; half maximal rates were estimated at 0.1 mM CO2+H2CO3. Under saturating conditions of light and CO2, 14CO2 was fixed primarily into 3-PGA, and subsequently moved into sugar phosphates and amino acids. Incorporation into aspartate was relatively slow.CO2 fixation was strictly light-dependent. The changes in adenylate and pyridine nucleotide pools were followed in light/dark and dark/light transitions. Whereas adenylates relaxed slowly over 15–20 min to the concentrations characteristic of illuminated cells following the abrupt changes induced by darkening, the sharp drop in intracellular NADPH showed little dark recovery although rapid restoration occurred on reillumination. Other pyridine nucleotides showed no changes during these transitions. The nucleotide specificity and Km of partially purified GAP dehydrogenase suggest a role for this enzyme in the regulation of CO2 fixation.

Chloroherpeton thalassium gen. nov. et spec. nov., a non-filamentous, flexing and gliding green sulfur bacterium.

A flexing and gliding green sulfur bacterium has been isolated from marine sources off the North East coast of the USA. Chloroherpeton thalassium is an obligate phototroph, and requires CO2 and S2- for growth; some organic acids can contribute to cell carbon, and N2 may be fixed. The cells contain typical chlorosomes, and gas vesicles may be present. Bacteriochlorophyll c is the main light harvesting pigment, and a small quantity of bacteriochlorophyll a is also present. Over 80% of the carotenoid is γ-carotene. DNA base composition of the isolates ranges from 45.0–48.2 mol% G+C.

Reductive, Coenzyme A-Mediated Pathway for 3-Chlorobenzoate Degradation in the Phototrophic Bacterium Rhodopseudomonas palustris

ABSTRACT We isolated a strain of Rhodopseudomonas palustris(RCB100) by selective enrichment in light on 3-chlorobenzoate to investigate the steps that it uses to accomplish anaerobic dechlorination. Analyses of metabolite pools as well as enzyme assays suggest that R. palustris grows on 3-chlorobenzoate by (i) converting it to 3-chlorobenzoyl coenzyme A (3-chlorobenzoyl–CoA), (ii) reductively dehalogenating 3-chlorobenzoyl–CoA to benzoyl-CoA, and (iii) degrading benzoyl-CoA to acetyl-CoA and carbon dioxide.R. palustris uses 3-chlorobenzoate only as a carbon source and thus incorporates the acetyl-CoA that is produced into cell material. The reductive dechlorination route used by R. palustris for 3-chlorobenzoate degradation differs from those previously described in that a CoA thioester, rather than an unmodified aromatic acid, is the substrate for complete dehalogenation.

Permeability of ammonia, methylamine and ethylamine in the cyanobacterium,Synechococcus R-2 (Anacystis nidulans) PCC 7942

SummaryPermeabilities of ammonia (NH3), methylamine (CH3NH2) and ethylamine (CH3CH2NH2) in the cyanobacterium (cyanophyte)Synechococcus R-2 (Anacystis nidulans) have been measured. Based on net uptake rates of DCMU (dichlorophenyldimethylurea) treated cells, the permeability of ammonia was 6.44±1.22 μm sec−1 (n=13). The permeabilities of methylamine and ethylamine, based on steady-state14C labeling were more than ten times that of ammonia (Pmethylamine=84.6±9.47 μm sec−1 (76),Pethylamine=109±11 μm sec−1 (55)). The apparent permeabilities based on net uptake rates of methylamine and ethylamine uptake were significantly lower, but this effect was partially reversible by ammonia, suggesting that net amine fluxes are rate limited by proton fluxes to an upper limit of about 700 nmol m−2 sec−1. Increasing concentrations of amines in alkaline conditions partially dissipated the pH gradient across the cell membrane, and this property could be used to calculate the relative permeabilities of different amines. The ratio of ethylamine to methylamine permeabilities was not significantly different from that calculated from the direct measurements of permeabilities; ammonia was much less effective in dissipating the pH gradient across the cell membrane than methylamine or ethylamine. An apparent permeability of ammonia of 5.7±0.9 μm sec−1 could be calculated from the permeability ratio of ammonia to methylamine and the experimentally measured permeability of methylamine. The permeability properties of ammonia and methylamine are very different; this poses problems in the interpretation of experiments where14C-methylamine is used as an ammonia analogue.