Caroline S. Harwood

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.

Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor.

High levels of the intracellular signalling molecule cyclic diguanylate (c‐di‐GMP) supress motility and activate exopolysaccharide (EPS) production in a variety of bacterial species. In many bacteria part of the effect of c‐di‐GMP is on gene expression, but the mechanism involved is not known for any species. We have identified the protein FleQ as a c‐di‐GMP‐responsive transcriptional regulator in Pseudomonas aeruginosa. FleQ is known to activate expression of flagella biosynthesis genes. Here we show that it also represses transcription of genes including the pel operon involved in EPS biosynthesis, and that this repression is relieved by c‐di‐GMP. Our in vivo data indicate that FleQ represses pel transcription and that pel transcription is not repressed when intracellular c‐di‐GMP levels are high. FleN, a known antiactivator of FleQ also participates in control of pel expression. In in vitro experiments we found that FleQ binds to pel promoter DNA and that this binding is inhibited by c‐di‐GMP. FleQ binds radiolabelled c‐di‐GMP in vitro. FleQ does not have amino acid motifs that resemble previously defined c‐di‐GMP binding domains. Our results show that FleQ is a new type of c‐di‐GMP binding protein that controls the transcriptional regulation of EPS biosynthesis genes in P. aeruginosa.

The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing

In Escherichia coli and some other γ‐Proteobacteria, the alternative σ factor RpoS functions as a regulator of the general stress response. The role of RpoS in Pseudomonas aeruginosa is not clear. Although P. aeruginosa RpoS contributes to the resistance to several environmental stresses, its role appears to be less pivotal than in E. coli. In P. aeruginosa, RpoS also regulates the production of several virulence factors and influences the expression of individual genes that are controlled by quorum sensing. Some quorum‐controlled genes are induced by RpoS, whereas others are repressed. To gain insights about RpoS function in P. aeruginosa and to understand better the regulation of quorum‐controlled genes, we used transcript profiling to define an RpoS regulon. We identified 772 genes regulated by RpoS in stationary but not in logarithmic growth phase (504 were induced and 268 were repressed), and we identified putative RpoS promoter sequence elements with similarity to the E. coli RpoS consensus in several of these genes. Many genes in the regulon, for example a set of chemotaxis genes, have assigned functions that are distinct from those in E. coli and are not obviously related to a stress response. Furthermore, RpoS affects the expression of more than 40% of all quorum‐controlled genes identified in our previous transcriptome analysis. This highlights the significance of RpoS as a global factor that controls quorum‐sensing gene expression at the onset of stationary phase. The transcription profiling results have allowed us to build a model that accommodates previous seemingly conflicting reports.

A new class of homoserine lactone quorum-sensing signals

Quorum sensing is a term used to describe cell-to-cell communication that allows cell-density-dependent gene expression. Many bacteria use acyl-homoserine lactone (acyl-HSL) synthases to generate fatty acyl-HSL quorum-sensing signals, which function with signal receptors to control expression of specific genes. The fatty acyl group is derived from fatty acid biosynthesis and provides signal specificity, but the variety of signals is limited. Here we show that the photosynthetic bacterium Rhodopseudomonas palustris uses an acyl-HSL synthase to produce p-coumaroyl-HSL by using environmental p-coumaric acid rather than fatty acids from cellular pools. The bacterium has a signal receptor with homology to fatty acyl-HSL receptors that responds to p-coumaroyl-HSL to regulate global gene expression. We also found that p-coumaroyl-HSL is made by other bacteria including Bradyrhizobium sp. and Silicibacter pomeroyi. This discovery extends the range of possibilities for acyl-HSL quorum sensing and raises fundamental questions about quorum sensing within the context of environmental signalling.

Pseudomonas aeruginosa Rugose Small-Colony Variants Have Adaptations That Likely Promote Persistence in the Cystic Fibrosis Lung

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.

Responses of Pseudomonas aeruginosa to low oxygen indicate that growth in the cystic fibrosis lung is by aerobic respiration

Pseudomonas aeruginosa in the lungs of cystic fibrosis patients grows to high densities in mucopurulent material that is depleted in oxygen. Some have concluded that growth in these circumstances is dependent on anaerobic nitrate respiration. Here we present data in favour of the alternative hypothesis that microaerobic respiration is the predominant mode of P. aeruginosa growth in the cystic fibrosis lung. We found that P. aeruginosa strain PAO1 and a mucoid derivative of strain PAO1 each grew at dissolved oxygen concentrations of less than 3 μM. This is lower than the concentration of oxygen that has been measured in hypoxic cystic fibrosis mucous. A transcriptome analysis comparing cells grown under aerobic conditions (185 μM dissolved oxygen) with cells grown with 20 μM or 3 μM dissolved oxygen, or anaerobically with nitrate, revealed that overlapping sets of genes are expressed depending on oxygen availability. This suggests that P. aeruginosa responds to changes in oxygen concentration along a continuum rather than having a discrete low oxygen regulon. Any one of three high affinity terminal oxidases that P. aeruginosa encodes supported microaerobic growth. A triple mutant lacking all three of these oxidases failed to grow at low oxygen and formed abnormal biofilms.

Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene.

ABSTRACT The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1,Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putidaF1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.

Subcellular location characteristics of the Pseudomonas aeruginosa GGDEF protein, WspR, indicate that it produces cyclic-di-GMP in response to growth on surfaces

The Pseudomonas aeruginosa Wsp signal transduction system produces cyclic‐di‐GMP (c‐di‐GMP), an intracellular messenger that stimulates biofilm formation and suppresses motility. The Wsp system is homologous to chemotaxis systems and includes a membrane‐bound receptor protein, WspA, and a response regulator GGDEF protein, WspR, that catalyses c‐di‐GMP synthesis when phosphorylated. We found that the subcellular distributions of fluorescent protein‐tagged WspA and WspR differed markedly from their chemotaxis counterparts. WspA–YFP formed patches in cells whereas WspR–YFP was dispersed when unphosphorylated and formed bright cytoplasmic clusters when phosphorylated. WspR formed clusters in cells of a ΔwspF mutant, a genetic background that causes constitutive phosphorylation of WspR, but was dispersed in cells of a wspA mutant, a genetic background necessary for WspR phosphorylation. In addition, WspR mutated at Asp70, its predicted site of phosphorylation, did not form clusters. C‐di‐GMP synthesis was not required for cluster formation. WspR–YFP was dispersed in liquid‐grown wild‐type cells, but formed clusters that sometimes appeared and disappeared over the course of a few minutes in cells grown on an agar surface. Our results suggest that the compartmentalized production of c‐di‐GMP in response to a stimulus associated with growth on a surface is an important functional characteristic of the Wsp system.

Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria

The Calvin-Benson-Bassham cycle (Calvin cycle) catalyzes virtually all primary productivity on Earth and is the major sink for atmospheric CO2. A less appreciated function of CO2 fixation is as an electron-accepting process. It is known that anoxygenic phototrophic bacteria require the Calvin cycle to accept electrons when growing with light as their sole energy source and organic substrates as their sole carbon source. However, it was unclear why and to what extent CO2 fixation is required when the organic substrates are more oxidized than biomass. To address these questions we measured metabolic fluxes in the photosynthetic bacterium Rhodopseudomonas palustris grown with 13C-labeled acetate. R. palustris metabolized 22% of acetate provided to CO2 and then fixed 68% of this CO2 into cell material using the Calvin cycle. This Calvin cycle flux enabled R. palustris to reoxidize nearly half of the reduced cofactors generated during conversion of acetate to biomass, revealing that CO2 fixation plays a major role in cofactor recycling. When H2 production via nitrogenase was used as an alternative cofactor recycling mechanism, a similar amount of CO2 was released from acetate, but only 12% of it was reassimilated by the Calvin cycle. These results underscore that N2 fixation and CO2 fixation have electron-accepting roles separate from their better-known roles in ammonia production and biomass generation. Some nonphotosynthetic heterotrophic bacteria have Calvin cycle genes, and their potential to use CO2 fixation to recycle reduced cofactors deserves closer scrutiny.

Bacterial chemotaxis to pollutants and plant-derived aromatic molecules.

There is accumulating evidence that motile bacteria are chemotactically attracted to environmental pollutants that they can degrade. Chemotaxis, the ability of motile bacteria to detect and respond to specific chemicals in the environment, can increase an organisms chances of locating useful sources of carbon, nitrogen and energy, and could thus play an important role in the biodegradation process. Recent evidence demonstrating that chemotaxis and biodegradation genes are coordinately regulated suggests that these processes are intimately linked in nature.