Regulation of polyphosphate kinase production by antisense RNA in Pseudomonas fluorescens Pf0-1.

Pseudomonas spp. adapt rapidly to environmental fluctuations. Loss or overproduction of polyphosphate reduces the fitness of Pseudomonas fluorescens Pf0-1, indicating the importance of the fine-tuning of polyphosphate production. An antisense RNA was investigated and shown to regulate the polyphosphate kinase gene (ppk) by a posttranscriptional mechanism reducing ppk transcript abundance.

Novel genes involved in Pseudomonas fluorescens Pf0-1 motility and biofilm formation.

AdnA in Pseudomonas fluorescens, an ortholog of FleQ in P. aeruginosa, regulates both motility and flagellum-mediated attachment to various surfaces. A whole-genome microarray determined the AdnA transcriptome by comparing the gene expression pattern of wild-type Pf0-1 to that of Pf0-2x (adnA deletion mutant) in broth culture. In the absence of AdnA, expression of 92 genes was decreased, while 11 genes showed increased expression. Analysis of 16 of these genes fused to lacZ confirmed the microarray results. Several genes were further evaluated for their role in motility and biofilm formation. Two genes, Pfl01_1508 and Pfl01_1517, affected motility and had different effects on biofilm formation in Pf0-1. These two genes are predicted to specify proteins similar to the glycosyl transferases FgtA1 and FgtA2, which have been shown to be involved in virulence and motility in P. syringae. Three other genes, Pfl01_1516, Pfl01_1572, and Pfl01_1573, not previously associated with motility and biofilm formation in Pseudomonas had similar effects on biofilm formation in Pf0-1. Deletion of each of these genes led to different motility defects. Our data revealed an additional level of complexity in the control of flagellum function beyond the core genes known to be required and may yield insights into processes important for environmental persistence of P. fluorescens Pf0-1.

Extracellular electron transfer via microbial nanowires.

Microbes that can transfer electrons to extracellular electron acceptors, such as Fe(iii) oxides, are important in organic matter degradation and nutrient cycling in soils and sediments. Previous investigations on electron transfer to Fe(iii) have focused on the role of outer-membrane c-type cytochromes. However, some Fe(iii) reducers lack c-cytochromes. Geobacter species, which are the predominant Fe(iii) reducers in many environments, must directly contact Fe(iii) oxides to reduce them, and produce monolateral pili that were proposed, on the basis of the role of pili in other organisms, to aid in establishing contact with the Fe(iii) oxides. Here we report that a pilus-deficient mutant of Geobacter sulfurreducens could not reduce Fe(iii) oxides but could attach to them. Conducting-probe atomic force microscopy revealed that the pili were highly conductive. These results indicate that the pili of G. sulfurreducens might serve as biological nanowires, transferring electrons from the cell surface to the surface of Fe(iii) oxides. Electron transfer through pili indicates possibilities for other unique cell-surface and cell-cell interactions, and for bioengineering of novel conductive materials.

Requirement of polyphosphate by Pseudomonas fluorescens Pf0-1 for competitive fitness and heat tolerance in laboratory media and sterile soil.

Knowledge of the genetic basis for bacterial survival and persistence in soil is a critical component in the development of successful biological control strategies and for understanding the ecological success of bacteria. We found a locus specifying polyphosphate kinase (ppk) and a nonpredicted antisense RNA (iiv8) in Pseudomonas fluorescens Pf0-1 to be necessary for optimal competitive fitness in LB broth culture and sterile loam soil. Pf0-1 lacking ppk and iiv8 was more than 10-fold less competitive against wild-type Pf0-1 in sterile loam soil low in inorganic phosphate. Studies indicated that ppk, and not iiv8, was required for competitive fitness. No role for iiv8 was identified. While a ppk and iiv8 mutant of Pf0-1 did not have increased sensitivity to osmotic, oxidative, and acid stress, it was more sensitive to elevated temperatures in laboratory medium and during growth in sterile soil. ppk was shown to be part of the Pho regulon in P. fluorescens, being upregulated in response to a low external P(i) concentration. Of importance, overproduction of polyphosphate in the soil environment appears to be more deleterious than production of none at all. Our findings reveal a new role for polyphosphate (and the need for proper regulation of its production) in competitive fitness of P. fluorescens in laboratory and soil environments.

Proteomic detection of non-annotated protein-coding genes in Pseudomonas fluorescens Pf0-1.

Genome sequences are annotated by computational prediction of coding sequences, followed by similarity searches such as BLAST, which provide a layer of possible functional information. While the existence of processes such as alternative splicing complicates matters for eukaryote genomes, the view of bacterial genomes as a linear series of closely spaced genes leads to the assumption that computational annotations that predict such arrangements completely describe the coding capacity of bacterial genomes. We undertook a proteomic study to identify proteins expressed by Pseudomonas fluorescens Pf0-1 from genes that were not predicted during the genome annotation. Mapping peptides to the Pf0-1 genome sequence identified sixteen non-annotated protein-coding regions, of which nine were antisense to predicted genes, six were intergenic, and one read in the same direction as an annotated gene but in a different frame. The expression of all but one of the newly discovered genes was verified by RT-PCR. Few clues as to the function of the new genes were gleaned from informatic analyses, but potential orthologs in other Pseudomonas genomes were identified for eight of the new genes. The 16 newly identified genes improve the quality of the Pf0-1 genome annotation, and the detection of antisense protein-coding genes indicates the under-appreciated complexity of bacterial genome organization.

Possible nonconductive role of Geobacter sulfurreducens pilus nanowires in biofilm formation.

Geobacter sulfurreducens required expression of electrically conductive pili to form biofilms on Fe(III) oxide surfaces, but pili were also essential for biofilm development on plain glass when fumarate was the sole electron acceptor. Furthermore, pili were needed for cell aggregation in agglutination studies. These results suggest that the pili of G. sulfurreducens also have a structural role in biofilm formation.

Subsurface clade of Geobacteraceae that predominates in a diversity of Fe(III)-reducing subsurface environments.

There are distinct differences in the physiology of Geobacter species available in pure culture. Therefore, to understand the ecology of Geobacter species in subsurface environments, it is important to know which species predominate. Clone libraries were assembled with 16S rRNA genes and transcripts amplified from three subsurface environments in which Geobacter species are known to be important members of the microbial community: (1) a uranium-contaminated aquifer located in Rifle, CO, USA undergoing in situ bioremediation; (2) an acetate-impacted aquifer that serves as an analog for the long-term acetate amendments proposed for in situ uranium bioremediation and (3) a petroleum-contaminated aquifer in which Geobacter species play a role in the oxidation of aromatic hydrocarbons coupled with the reduction of Fe(III). The majority of Geobacteraceae 16S rRNA sequences found in these environments clustered in a phylogenetically coherent subsurface clade, which also contains a number of Geobacter species isolated from subsurface environments. Concatamers constructed with 43 Geobacter genes amplified from these sites also clustered within this subsurface clade. 16S rRNA transcript and gene sequences in the sediments and groundwater at the Rifle site were highly similar, suggesting that sampling groundwater via monitoring wells can recover the most active Geobacter species. These results suggest that further study of Geobacter species in the subsurface clade is necessary to accurately model the behavior of Geobacter species during subsurface bioremediation of metal and organic contaminants.

Biofilm and nanowire production leads to increased current in Geobacter sulfurreducens fuel cells.

Geobacter sulfurreducens developed highly structured, multilayer biofilms on the anode surface of a microbial fuel cell converting acetate to electricity. Cells at a distance from the anode remained viable, and there was no decrease in the efficiency of current production as the thickness of the biofilm increased. Genetic studies demonstrated that efficient electron transfer through the biofilm required the presence of electrically conductive pili. These pili may represent an electronic network permeating the biofilm that can promote long-range electrical transfer in an energy-efficient manner, increasing electricity production more than 10-fold.

Potential role of a novel psychrotolerant member of the family Geobacteraceae, Geopsychrobacter electrodiphilus gen. nov., sp. nov., in electricity production by a marine sediment fuel cell.

Previous studies have shown that members of the family Geobacteraceae that attach to the anodes of sediment fuel cells are directly involved in harvesting electricity by oxidizing organic compounds to carbon dioxide and transferring the electrons to the anode. In order to learn more about this process, microorganisms from the anode surface of a marine sediment fuel cell were enriched and isolated with Fe(III) oxide. Two unique marine isolates were recovered, strains A1(T) and A2. They are gram-negative, nonmotile rods, with abundant c-type cytochromes. Phylogenetic analysis of the 16S rRNA, recA, gyrB, fusA, rpoB, and nifD genes indicated that strains A1(T) and A2 represent a unique phylogenetic cluster within the Geobacteraceae. Both strains were able to grow with an electrode serving as the sole electron acceptor and transferred ca. 90% of the electrons available in their organic electron donors to the electrodes. These organisms are the first psychrotolerant members of the Geobacteraceae reported thus far and can grow at temperatures between 4 and 30 degrees C, with an optimum temperature of 22 degrees C. Strains A1(T) and A2 can utilize a wide range of traditional electron acceptors, including all forms of soluble and insoluble Fe(III) tested, anthraquinone 2,6-disulfonate, and S(0). In addition to acetate, both strains can utilize a number of other organic acids, amino acids, long-chain fatty acids, and aromatic compounds to support growth with Fe(III) nitrilotriacetic acid as an electron acceptor. The metabolism of these organisms differs in that only strain A1(T) can use acetoin, ethanol, and hydrogen as electron donors, whereas only strain A2 can use lactate, propionate, and butyrate. The name Geopsychrobacter electrodiphilus gen. nov., sp. nov., is proposed for strains A1(T) and A2, with strain A1(T) (ATCC BAA-880(T); DSM 16401(T); JCM 12469) as the type strain. Strains A1(T) and A2 (ATCC BAA-770; JCM 12470) represent the first organisms recovered from anodes that can effectively couple the oxidation of organic compounds to an electrode. Thus, they may serve as important model organisms for further elucidation of the mechanisms of microbe-electrode electron transfer in sediment fuel cells.