Increased fitness of Pseudomonas fluorescens Pf0-1 leucine auxotrophs in soil

The annotation process of a newly sequenced bacterial genome is largely based on algorithms derived from databases of previously defined RNA and protein-encoding gene structures. This process generally excludes the possibility that the two strands of a given stretch of DNA can each harbor a gene in an overlapping manner. While the presence of such structures in eukaryotic genomes is considered to be relatively common, their counterparts in prokaryotic genomes are just beginning to be recognized. Application of an in vivo expression technology has previously identified 22 discrete genetic loci in Pseudomonas fluorescens Pf0-1 that were specifically activated in the soil environment, of which 10 were present in an antisense orientation relative to previously annotated genes. This observation led to the hypothesis that the physiological role of overlapping genetic structures may be relevant to growth conditions outside artificial laboratory media. Here, we examined the role of one of the overlapping gene pairs, iiv19 and leuA2, in soil. Although iiv19 was previously demonstrated to be preferentially activated in the soil environment, its absence did not alter the ability of P. fluorescens to colonize or survive in soil. Surprisingly, the absence of the leuA2 gene conferred a fitness advantage in the soil environment when leucine was supplied exogenously. This effect was determined to be independent of the iiv19 gene, and further analyses revealed that amino acid antagonism was the underlying mechanism behind the observed fitness advantage of the bacterium in soil. Our findings provide a potential mechanism for the frequent occurrence of auxotrophic mutants of Pseudomonas spp. in the lungs of cystic fibrosis patients.     

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.     

No apparent costs for facultative antibiotic production by the soil bacterium Pseudomonas fluorescens Pf0-1

Background

Many soil-inhabiting bacteria are known to produce secondary metabolites that can suppress microorganisms competing for the same resources. The production of antimicrobial compounds is expected to incur fitness costs for the producing bacteria. Such costs form the basis for models on the co-existence of antibiotic-producing and non-antibiotic producing strains. However, so far studies quantifying the costs of antibiotic production by bacteria are scarce. The current study reports on possible costs, for antibiotic production by Pseudomonas fluorescens Pf0-1, a soil bacterium that is induced to produce a broad-spectrum antibiotic when it is confronted with non-related bacterial competitors or supernatants of their cultures.

Methodology and Principal Findings

We measured the possible cost of antibiotic production for Pseudomonas fluorescens Pf0-1 by monitoring changes in growth rate with and without induction of antibiotic production by supernatant of a bacterial competitor, namely Pedobacter sp.. Experiments were performed in liquid as well as on semi-solid media under nutrient-limited conditions that are expected to most clearly reveal fitness costs. Our results did not reveal any significant costs for production of antibiotics by Pseudomonas fluorescens Pf0-1. Comparison of growth rates of the antibiotic-producing wild-type cells with those of non-antibiotic producing mutants did not reveal costs of antibiotic production either.

Significance

Based on our findings we propose that the facultative production of antibiotics might not be selected to mitigate metabolic costs, but instead might be advantageous because it limits the risk of competitors evolving resistance, or even the risk of competitors feeding on the compounds produced.     

Pleiotropic Effects of GacA on Pseudomonas fluorescens Pf0-1 In Vitro and in Soil

Pseudomonas species can exhibit phenotypic variation resulting from gacS or gacA mutation. P. fluorescens Pf0-1 is a gacA mutant and exhibits pleiotropic changes following the introduction of a functional allele. GacA enhances biofilm development while reducing dissemination in soil, suggesting that alternative Gac phenotypes enable Pseudomonas sp. to exploit varied environments.     

Spaceflight Promotes Biofilm Formation by Pseudomonas aeruginosa

Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight.     

假单胞菌Pseudomonas fluorescens Pf0-1中转录调控因子SpfB负调控DNA磷硫酰化修饰（英文）

【目的】DNA磷硫酰化修饰是DNA骨架上非桥接的氧原子以序列选择性和R-构型被硫取代的一种新型DNA修饰。目前,磷硫酰化修饰在多种细菌、古生菌以及人类致病菌中多有发现,但其分子调控机制尚不清楚。为了全面解析磷硫酰化修饰的调控机制,本文选择荧光假单胞菌Pf0-1为研究对象,开展了其DNA磷硫酰化修饰的调控机制研究。【方法】首先,构建了spfB基因缺失和回补菌株,使用碘能特异性断裂磷硫酰化修饰DNA的方法,研究了该基因缺失对修饰表型的影响。利用cDNA在相邻同方向的基因间隔区进行PCR,确定了磷硫酰化修饰基因簇spf BCDE内的共转录单元。通过荧光定量RT-PCR,分析了spfB基因缺失突变株中磷硫酰化修饰基因的转录量。利用异源表达并纯化得到的重组蛋白SpfB进行了体外功能研究。通过EMSA实验,验证了SpfB蛋白具有与spfB启动子序列结合活性。通过DNase I footprinting实验,精确定位了Spf B蛋白与DNA结合序列。【结果】spf B基因的缺失加剧了磷硫酰化修饰DNA断裂所致电泳条带弥散的表型,spf B基因的回补能够恢复该表型,证明spf B基因负调控磷硫酰化修饰。鉴定了spf基因簇中只含有1个共转录单元,且该共转录单元在?spfB突变株中转录水平明显上升。通过EMSA和DNase I footprint实验,检测了SpfB蛋白与磷硫酰化修饰基因spf BCDE的启动子区域5′-TGTTTGT-3′相结合。【结论】SpfB作为转录调控因子负调控磷硫酰化修饰基因spf BCDE的表达,为解析磷硫酰化修饰的调控机制和全面理解基因组上的部分修饰特征奠定了基础。     

Pseudomonas aeruginosa Promotes Escherichia coli Biofilm Formation in Nutrient-Limited Medium

Biofilms have been implicated as an important reservoir for pathogens and commensal enteric bacteria such as Escherichia coli in natural and engineered water systems. However, the processes that regulate the survival of E. coli in aquatic biofilms have not been thoroughly studied. We examined the effects of hydrodynamic shear and nutrient concentrations on E. coli colonization of pre-established Pseudomonas aeruginosa biofilms, co-inoculation of E. coli and P. aeruginosa biofilms, and P. aeruginosa colonization of pre-established E. coli biofilms. In nutritionally-limited R2A medium, E. coli dominated biofilms when co-inoculated with P. aeruginosa, and successfully colonized and overgrew pre-established P. aeruginosa biofilms. In more enriched media, P. aeruginosa formed larger clusters, but E. coli still extensively overgrew and colonized the interior of P. aeruginosa clusters. In mono-culture, E. coli formed sparse and discontinuous biofilms. After P. aeruginosa was introduced to these biofilms, E. coli growth increased substantially, resulting in patterns of biofilm colonization similar to those observed under other sequences of organism introduction, i.e., E. coli overgrew P. aeruginosa and colonized the interior of P. aeruginosa clusters. These results demonstrate that E. coli not only persists in aquatic biofilms under depleted nutritional conditions, but interactions with P. aeruginosa can greatly increase E. coli growth in biofilms under these experimental conditions.     

LapG, required for modulating biofilm formation by Pseudomonas fluorescens Pf0-1, is a calcium-dependent protease

Biofilm formation by Pseudomonas fluorescens Pf0-1 requires the cell surface adhesin LapA. We previously reported that LapG, a periplasmic cysteine protease of P. fluorescens, cleaves the N terminus of LapA, thus releasing this adhesin from the cell surface and resulting in loss of the ability to make a biofilm. The activity of LapG is regulated by the inner membrane-localized cyclic-di-GMP receptor LapD via direct protein-protein interactions. Here we present chelation and metal add-back studies demonstrating that calcium availability regulates biofilm formation by P. fluorescens Pf0-1. The determination that LapG is a calcium-dependent protease, based on in vivo and in vitro studies, explains the basis of this calcium-dependent regulation. Based on the crystal structure of LapG of Legionella pneumophila in the accompanying report by Chatterjee and colleagues (D. Chatterjee et al., J. Bacteriol. 194:4415-4425, 2012), we show that the calcium-binding residues of LapG, D134 and E136, which are near the critical C135 active-site residue, are required for LapG activity of P. fluorescens in vivo and in vitro. Furthermore, we show that mutations in D134 and E136 result in LapG proteins no longer able to interact with LapD, indicating that calcium binding results in LapG adopting a conformation competent for interaction with the protein that regulates its activity. Finally, we show that citrate, an environmentally relevant calcium chelator, can impact LapG activity and thus biofilm formation, suggesting that a physiologically relevant chelator of calcium can impact biofilm formation by this organism.     

Phosphate-dependent modulation of c-di-GMP levels regulates Pseudomonas fluorescens Pf0-1 biofilm formation by controlling secretion of the adhesin LapA

Biofilm formation is commonly described as a developmental process regulated by environmental cues. In the current study we present a mechanistic model to explain regulation of Pseudomonas fluorescens biofilm formation by the environmentally relevant signal inorganic phosphate (P(i)). We show that activation of the Pho regulon, the major pathway for adaptation to phosphate limitation, results in conditional expression of a c-di-GMP phosphodiesterase referred to as RapA. Genetic analysis indicated that RapA is an inhibitor of biofilm formation and required for loss of biofilm formation in response to limiting P(i). Our results suggest that RapA lowers the level of c-di-GMP, which in turn inhibits the secretion of LapA, a large adhesion required for biofilm formation by P. fluorescens. The ability of c-di-GMP to modulate protein secretion is a novel finding and further extends the biological influence of c-di-GMP beyond that of regulating exopolysaccharide synthesis and motility. Interestingly, Pho regulon expression does not impinge on the rate of attachment to a surface but rather inhibits the transition of cells to a more stable interaction with the surface. We hypothesize that Pho regulon expression confers a surface-sensing mode on P. fluorescens and suggest this strategy may be broadly applicable to other bacteria.     

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.Soils are complex environments, presenting microbial inhabitants with a range of challenges which must be met if they are to survive and persist. Nutrients, pH, water content, and temperature can all affect survival of bacteria in soil (44). An understanding of the ecological success of microbes in natural environments such as soil requires knowledge of the mechanisms underlying adaptation and persistence. This appreciation for adaptive mechanisms is also important for the successful use of microbes for environmental applications, such as biocontrol or bioremediation (29, 47). Pseudomonas species, which are frequently isolated from soil environments, have a large complement of regulatory genes which are thought to permit rapid responses to environmental changes (42). For example, the two-component pair phoB-phoR responds to change in the concentration of exogenous inorganic phosphate (P_i) (25, 46). The regulatory targets of such systems likely include genes that are critical for adaptation and thus are important for long-term success within a fluctuating environment.Many Pseudomonas species are being investigated as potential biocontrol agents because of their ability to produce compounds that are inhibitory to plant-pathogenic fungi (10). In addition, some Pseudomonas species have plant growth-promoting activity independent of their antifungal activity, making them very attractive in biocontrol (45). Despite the frequency with which Pseudomonas species are isolated from soils and the interest in developing biocontrol applications, the determinants of their environmental success are not well characterized (8).Genes that are upregulated in a particular environment are likely to be important for success in that environment (28). Indeed, a number of environmentally regulated genes, identified using in vivo expression technology (IVET), have been shown to be important for success in the environment from which they were isolated (for examples, see references 3, 8, 19, and 20). We have been investigating the genetic basis of persistence of the soil isolate Pseudomonas fluorescens Pf0-1. Using an IVET screen, we found 22 sequences upregulated during growth in sterile loam soil (40). Three of these IVET-identified genes were shown to be involved in colonization of sterile soil. Here we report the analysis of an additional IVET-identified locus, termed iiv8 (40). The sequence upregulated in soil did not match any gene predicted in the Pf0-1 genome annotation (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000094","term_id":"253992019","term_text":"CP000094"}}CP000094) and was antisense to a predicted polyphosphate (poly P) kinase gene (Pfl01_5464). In a previous study, we demonstrated that a nonpredicted IVET-identified antisense gene specified a protein of importance in colonization of sterile soil (39). Kim and Levy (16) recently showed that at a sense/antisense locus, at which the antisense gene was identified by IVET, the sense gene had a role in soil fitness.poly P is a polymer of inorganic phosphate found ubiquitously and is important for a number of processes in bacteria (reviewed in reference 36). For example, in Escherichia coli, loss of poly P production is associated with defects in stationary-phase survival and with tolerance of osmotic, oxidative, and heat stress (30). In Vibrio cholerae poly P is important for motility (33), surface attachment (27), and tolerance of low pH and oxidative and osmotic stress (12). Proposed functions of poly P include chelation of metals, phosphate storage, substitution for ATP, and maintenance of pH (reviewed in reference 18). In Pseudomonas aeruginosa PAO1, poly P plays a role in numerous traits including motility (32); biofilm formation, quorum sensing, and virulence (34); and carbenicillin tolerance and maintenance of normal cellular ultrastructure (7). In E. coli, poly P production is part of the Pho regulon (1), a suite of genes upregulated in response to low phosphate by the response regulator PhoB. When P_i is limiting, PhoB is phosphorylated by PhoR and can then activate the Pho regulon. When P_i is in excess, PhoR-mediated phosphorylation of PhoB is prevented by an interaction with the Pst (phosphate-specific transport) system. This regulatory system is widely conserved and has been demonstrated to be present in P. fluorescens Pf0-1. The Pho regulon is constitutively derepressed in pst mutants because of the inability to prevent phosphorylation of PhoB. phoB mutants cannot activate the Pho regulon, and hence, Pho regulon genes are repressed under all conditions (25).The objective of this study was to determine the importance of the overlapping gene pair ppk and iiv8 in stress tolerance and fitness. Studies of bacteria in soil sought to extend our understanding of survival and fitness traits of soil bacteria.     

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions¹. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions^2,3.The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.     

Candida albicans Ethanol Stimulates Pseudomonas aeruginosa WspR-Controlled Biofilm Formation as Part of a Cyclic Relationship Involving Phenazines

In chronic infections, pathogens are often in the presence of other microbial species. For example, Pseudomonas aeruginosa is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF) and co-infections with Candida albicans are common. Here, we show that P. aeruginosa biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus C. albicans. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic-di-GMP (c-di-GMP), and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted P. aeruginosa colonization of CF airway epithelial cells. P. aeruginosa production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by C. albicans. Using a C. albicans adh1/adh1 mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of P. aeruginosa phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both P. aeruginosa and C. albicans has been associated with worse outcomes in cystic fibrosis.     

Ciprofloxacin-Eluting Nanofibers Inhibits Biofilm Formation by Pseudomonas aeruginosa and a Methicillin-Resistant Staphylococcus aureus

Pseudomonas aeruginosa and Staphylococcus aureus are commonly associated with hospital-acquired infections and are known to form biofilms. Ciprofloxacin (CIP), which is normally used to treat these infections, is seldom effective in killing cells in a biofilm. This is mostly due to slow or weak penetration of CIP to the core of biofilms. The problem is accentuated by the release of CIP below MIC (minimal inhibitory concentration) levels following a rapid (burst) release. The aim of this study was to develop a drug carrier that would keep CIP above MIC levels for an extended period. Ciprofloxacin was suspended into poly(D,L-lactide) (PDLLA) and poly(ethylene oxide) (PEO), and electrospun into nanofibers (CIP-F). All of the CIP was released from the nanofibers within 2 h, which is typical of a burst release. However, 99% of P. aeruginosa PA01 cells and 91% of S. aureus Xen 30 cells (a methicillin-resistant strain) in biofilms were killed when exposed to CIP-F. CIP levels remained above MIC for 5 days, as shown by growth inhibition of the cells in vitro. The nanofibers were smooth in texture with no bead formation, as revealed by scanning electron and atomic force microscopy. A single vibration peak at 1632 cm^-1, recorded with Fourier transform infrared spectroscopy, indicated that CIP remained in crystal form when incorporated into PDLLA: PEO. No abnormalities in the histology of MCF-12A breast epithelial cells were observed when exposed to CIP-F. This is the first report of the inhibition of biofilm formation by CIP released from PDLLA: PEO nanofibers.