A reversible mutation in a genomic hotspot saves bacterial swarms from extinction.

Microbial adaptation to changing environmental conditions is frequently mediated by hypermutable sequences. Here we demonstrate that such a hypermutable hotspot within a gene encoding a flagellar unit of Paenibacillus glucanolyticus generated spontaneous non-swarming mutants with increased stress resistance. These mutants, which survived conditions that eliminated wild-type cultures, could be carried by their swarming siblings when the colony spread, consequently increasing their numbers at the spreading edge. Of interest, the hypermutable nature of the aforementioned sequence enabled the non-swarming mutants to serve as "seeds" for a new generation of wild-type cells through reversion of the mutation. Using a mathematical model, we examined the survival dynamics of P. glucanolyticus colonies under fluctuating environments. Our experimental and theoretical results suggest that the non-swarming, stress-resistant mutants can save the colony from extinction. Notably, we identified this hypermutable sequence in flagellar genes of additional Paenibacillus species, suggesting that this phenomenon could be wide-spread and ecologically important.

Bacterial-induced pH shifts link individual cell physiology to macroscale collective behavior.

Bacteria have evolved a diverse array of signaling pathways that enable them to quickly respond to environmental changes. Understanding how these pathways reflect environmental conditions and produce an orchestrated response is an ongoing challenge. Herein, we present a role for collective modifications of environmental pH carried out by microbial colonies living on a surface. We show that by collectively adjusting the local pH value, Paenibacillus spp., specifically, regulate their swarming motility. Moreover, we show that such pH-dependent regulation can converge with the carbon repression pathway to down-regulate flagellin expression and inhibit swarming in the presence of glucose. Interestingly, our results demonstrate that the observed glucose-dependent swarming repression is not mediated by the glucose molecule per se, as commonly thought to occur in carbon repression pathways, but rather is governed by a decrease in pH due to glucose metabolism. In fact, modification of the environmental pH by neighboring bacterial species could override this glucose-dependent repression and induce swarming of Paenibacillus spp. away from a glucose-rich area. Our results suggest that bacteria can use local pH modulations to reflect nutrient availability and link individual bacterial physiology to macroscale collective behavior.

Potential Control of Potato Soft Rot Disease by the Obligate Predators Bdellovibrio and Like Organisms.

Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. affect a wide range of crops, including potatoes, a major food crop. As of today, farmers mostly rely on sanitary practices, water management, and plant nutrition for control. We tested the bacterial predators Bdellovibrio and like organisms (BALOs) to control potato soft rot. BALOs are small, motile predatory bacteria found in terrestrial and aquatic environments. They prey on a wide range of Gram-negative bacteria, including animal and plant pathogens. To this end, BALO strains HD100, 109J, and a ΔmerRNA derivative of HD100 were shown to efficiently prey on various rot-causing strains of Pectobacterium and Dickeya solani BALO control of maceration caused by a highly virulent strain of Pectobacterium carotovorum subsp. brasilense was then tested in situ using a potato slice assay. All BALO strains were highly effective at reducing disease, up to complete prevention. Effectivity was concentration dependent, and BALOs applied before P. carotovorum subsp. brasilense inoculation performed significantly better than those applied after the disease-causing agent, maybe due to in situ consumption of glucose by the prey, as glucose metabolism by live prey bacteria was shown to prevent predation. Dead predators and the supernatant of BALO cultures did not significantly prevent maceration, indicating that predation was the major mechanism for the prevention of the disease. Finally, plastic resistance to predation was affected by prey and predator population parameters, suggesting that population dynamics affect prey response to predation.IMPORTANCE Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. are among the most important plant diseases caused by bacteria. Among other crops, they inflict large-scale damage to potatoes. As of today, farmers have few options to control them. The bacteria Bdellovibrio and like organisms (BALOs) are obligate predators of bacteria. We tested their potential to prey on Pectobacterium spp. and Dickeya spp. and to protect potato. We show that different BALOs can prey on soft rot-causing bacteria and prevent their growth in situ, precluding tissue maceration. Dead predators and the supernatant of BALO cultures did not significantly prevent maceration, showing that the effect is due to predation. Soft rot control by the predators was concentration dependent and was higher when the predator was inoculated ahead of the prey. As residual prey remained, we investigated what determines their level and found that initial prey and predator population parameters affect prey response to predation.

Smelling Pseudomonas aeruginosa infections using a whole-cell biosensor - An alternative for the gold-standard culturing assay.

Improved easy-to-use diagnostic tools for infections are in strong demand worldwide. Yet, despite dramatic advances in diagnostic technologies, the gold-standard remains culturing. Here we offer an alternative tool demonstrating that a bacterial biosensor can efficiently detect Pseudomonas aeruginosa infections in patients suffering from otitis externa. Detection was based on specific binding between the biosensor and 2-aminoacetophenone (2-AA), a volatile produced by P. aeruginosa in high amounts. We collected pus samples from ears of 26 subjects exhibiting symptoms of otitis externa. Detection of P. aeruginosa using the biosensor was compared to detection using gold-standard culturing assay and to gas-chromatograph-mass-spectrometry (GC-MS) analyses of 2-AA. The biosensor strain test matched the culture assay in 24 samples (92%) and the GC-MS analyses in 25 samples (96%). With this result in hand, we designed a device containing a whole-cell luminescent biosensor combined with a photo-multiplier tube. This device allowed detection of 2-AA at levels as low as 2 nmol, on par with detection level of GC-MS. The results of the described study demonstrate that the volatile 2-AA serves as an effective biomarker for P. aeruginosa in ear infections, and that activation of the biosensor strain by 2-AA provides a unique opportunity to design an easy-to-use device that can specifically detect P. aeruginosa infections.

The vapB-vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin-Antitoxin Module.

Toxin-antitoxin systems are commonly found on plasmids and chromosomes of bacteria and archaea. These systems appear as biscystronic genes encoding a stable toxin and a labile antitoxin, which protects the cells from the toxin's activity. Under specific, mostly stressful conditions, the unstable antitoxin is degraded, the toxin becomes active and growth is arrested. Using genome analysis we identified a putative toxin-antitoxin encoding system in the genome of the plant pathogen Acidovorax citrulli. The system is homologous to vapB-vapC systems from other bacterial species. PCR and phylogenetic analyses suggested that this locus is unique to group II strains of A. citrulli. Using biochemical and molecular analyses we show that A. citrulli VapBC module is a bona-fide toxin-antitoxin module in which VapC is a toxin with ribonuclease activity that can be counteracted by its cognate VapB antitoxin. We further show that transcription of the A. citrulli vapBC locus is induced by amino acid starvation, chloramphenicol and during plant infection. Due to the possible role of TA systems in both virulence and dormancy of human pathogenic bacteria, studies of these systems are gaining a lot of attention. Conversely, studies characterizing toxin-antitoxin systems in plant pathogenic bacteria are lacking. The study presented here validates the activity of VapB and VapC proteins in A. citrulli and suggests their involvement in stress response and host-pathogen interactions.

Silencing the mob: disrupting quorum sensing as a means to fight plant disease.

Bacteria are able to sense their population's density through a cell-cell communication system, termed 'quorum sensing' (QS). This system regulates gene expression in response to cell density through the constant production and detection of signalling molecules. These molecules commonly act as auto-inducers through the up-regulation of their own synthesis. Many pathogenic bacteria, including those of plants, rely on this communication system for infection of their hosts. The finding that the countering of QS-disrupting mechanisms exists in many prokaryotic and eukaryotic organisms offers a promising novel method to fight disease. During the last decade, several approaches have been proposed to disrupt QS pathways of phytopathogens, and hence to reduce their virulence. Such studies have had varied success in vivo, but most lend promising support to the idea that QS manipulation could be a potentially effective method to reduce bacterial-mediated plant disease. This review discusses the various QS-disrupting mechanisms found in both bacteria and plants, as well as the different approaches applied artificially to interfere with QS pathways and thus protect plant health.

Surface-motility induction, attraction and hitchhiking between bacterial species promote dispersal on solid surfaces.

The ability to move on solid surfaces provides ecological advantages for bacteria, yet many bacterial species lack this trait. We found that Xanthomonas spp. overcome this limitation by making use of proficient motile bacteria in their vicinity. Using X. perforans and Paenibacillus vortex as models, we show that X. perforans induces surface motility, attracts proficient motile bacteria and 'rides' them for dispersal. In addition, X. perforans was able to restore surface motility of strains that lost this mode of motility under multiple growth cycles in the lab. The described interaction occurred both on agar plates and tomato leaves and was observed between several xanthomonads and motile bacterial species. Thus, suggesting that this motility induction and hitchhiking strategy might be widespread and ecologically important. This study provides an example as to how bacteria can rely on the abilities of their neighboring species for their own benefit, signifying the importance of a communal organization for fitness.

Tri-party underground symbiosis between a weevil, bacteria and a desert plant.

Inhabitants of arid ecosystems face severe nitrogen and water limitations. Inventive adaptations by organisms occupying such habitats are essential for survival. This study describes a tri-party symbiotic interaction between a plant (Salsola inermis), a beetle (Conorhynchus pistor), and a bacterium (Klebsiella pneumonia). The weevil survives by living within a mud structure affixed to the plant roots, thus benefiting from increased carbon and water, and refuge from predators and parasites. Active nitrogen-fixing bacteria harbored within the weevil's gut mediate this interaction, by supplying nitrogen to the system, which eventually promotes seed development. We studied the correlation between the weevil's existence and (i) root carbon and nitrogen content, (ii) soil water content and (iii) seed weight. Roots hosting weevils contained more nitrogen, heavier seeds and less carbon. In addition, water content was higher around the roots than in open spaces a short distance from the plant stem. Bacterial studies and nitrogen-fixation analyses, including molecular and chemical assays, indicated atmospheric nitrogen fixation in the larval stage and identified the bacterium. The coexistence of weevil and bacterial behavior coinciding with the plant's life cycle was revealed here by a long period of field observations. Out of over 60,000 known weevils, this is the only report of a weevil living most of its life underground without harming plants. The unique tri-party interaction described herein shows the important ecological role of desert plant roots and provides an example of a sustainable consortium of living organisms coping with the challenging desert environment.

Identification and characterization of a highly motile and antibiotic refractory subpopulation involved in the expansion of swarming colonies of Paenibacillus vortex.

Bacteria often use sophisticated cooperative behaviours, such as the development of complex colonies, elaborate biofilms and advanced dispersal strategies, to cope with the harsh and variable conditions of natural habitats, including the presence of antibiotics. Paenibacillus vortex uses swarming motility and cell-to-cell communication to form complex, structured colonies. The modular organization of P. vortex colony has been found to facilitate its dispersal on agar surfaces. The current study reveals that the complex structure of the colony is generated by the coexistence and transition between two morphotypes--'builders' and 'explorers'--with distinct functions in colony formation. Here, we focused on the explorers, which are highly motile and spearhead colonial expansion. Explorers are characterized by high expression levels of flagellar genes, such as flagellin (hag), motA, fliI, flgK and sigD, hyperflagellation, decrease in ATP (adenosine-5'-triphosphate) levels, and increased resistance to antibiotics. Their tolerance to many antibiotics gives them the advantage of translocation through antibiotics-containing areas. This work gives new insights on the importance of cell differentiation and task distribution in colony morphogenesis and adaptation to antibiotics.

Genome sequence of the pattern-forming social bacterium Paenibacillus dendritiformis C454 chiral morphotype.

Paenibacillus dendritiformis is a Gram-positive, soil-dwelling, spore-forming social microorganism. An intriguing collective faculty of this strain is manifested by its ability to switch between different morphotypes, such as the branching (T) and the chiral (C) morphotypes. Here we report the 6.3-Mb draft genome sequence of the P. dendritiformis C454 chiral morphotype.

Genome sequence of the pattern forming Paenibacillus vortex bacterium reveals potential for thriving in complex environments.

BACKGROUND: The pattern-forming bacterium Paenibacillus vortex is notable for its advanced social behavior, which is reflected in development of colonies with highly intricate architectures. Prior to this study, only two other Paenibacillus species (Paenibacillus sp. JDR-2 and Paenibacillus larvae) have been sequenced. However, no genomic data is available on the Paenibacillus species with pattern-forming and complex social motility. Here we report the de novo genome sequence of this Gram-positive, soil-dwelling, sporulating bacterium. RESULTS: The complete P. vortex genome was sequenced by a hybrid approach using 454 Life Sciences and Illumina, achieving a total of 289× coverage, with 99.8% sequence identity between the two methods. The sequencing results were validated using a custom designed Agilent microarray expression chip which represented the coding and the non-coding regions. Analysis of the P. vortex genome revealed 6,437 open reading frames (ORFs) and 73 non-coding RNA genes. Comparative genomic analysis with 500 complete bacterial genomes revealed exceptionally high number of two-component system (TCS) genes, transcription factors (TFs), transport and defense related genes. Additionally, we have identified genes involved in the production of antimicrobial compounds and extracellular degrading enzymes. CONCLUSIONS: These findings suggest that P. vortex has advanced faculties to perceive and react to a wide range of signaling molecules and environmental conditions, which could be associated with its ability to reconfigure and replicate complex colony architectures. Additionally, P. vortex is likely to serve as a rich source of genes important for agricultural, medical and industrial applications and it has the potential to advance the study of social microbiology within Gram-positive bacteria.

Lethal protein produced in response to competition between sibling bacterial colonies.

Sibling Paenibacillus dendritiformis bacterial colonies grown on low-nutrient agar medium mutually inhibit growth through secretion of a lethal factor. Analysis of secretions reveals the presence of subtilisin (a protease) and a 12 kDa protein, termed sibling lethal factor (Slf). Purified subtilisin promotes the growth and expansion of P. dendritiformis colonies, whereas Slf is lethal and lyses P. dendritiformis cells in culture. Slf is encoded by a gene belonging to a large family of bacterial genes of unknown function, and the gene is predicted to encode a protein of approximately 20 kDa, termed dendritiformis sibling bacteriocin. The 20 kDa recombinant protein was produced and found to be inactive, but exposure to subtilisin resulted in cleavage to the active, 12 kDa form. The experimental results, combined with mathematical modeling, show that subtilisin serves to regulate growth of the colony. Below a threshold concentration, subtilisin promotes colony growth and expansion. However, once it exceeds a threshold, as occurs at the interface between competing colonies, Slf is then secreted into the medium to rapidly reduce cell density by lysis of the bacterial cells. The presence of genes encoding homologs of dendritiformis sibling bacteriocin in other bacterial species suggests that this mechanism for self-regulation of colony growth might not be limited to P. dendritiformis.

Extracellular matrix production and calcium carbonate precipitation by coral cells in vitro.

The evolution of multicellularity in animals required the production of extracellular matrices that serve to spatially organize cells according to function. In corals, three matrices are involved in spatial organization: (i) an organic ECM, which facilitates cell-cell and cell-substrate adhesion; (ii) a skeletal organic matrix (SOM), which facilitates controlled deposition of a calcium carbonate skeleton; and (iii) the calcium carbonate skeleton itself, which provides the structural support for the 3D organization of coral colonies. In this report, we examine the production of these three matrices by using an in vitro culturing system for coral cells. In this system, which significantly facilitates studies of coral cell physiology, we demonstrate in vitro excretion of ECM by primary (nondividing) tissue cultures of both soft (Xenia elongata) and hard (Montipora digitata) corals. There are structural differences between the ECM produced by X. elongata cell cultures and that of M. digitata, and ascorbic acid, a critical cofactor for proline hydroxylation, significantly increased the production of collagen in the ECM of the latter species. We further demonstrate in vitro production of SOM and extracellular mineralized particles in cell cultures of M. digitata. Inductively coupled plasma mass spectrometry analysis of Sr/Ca ratios revealed the particles to be aragonite. De novo calcification was confirmed by following the incorporation of (45)Ca into acid labile macromolecules. Our results demonstrate the ability of isolated, differentiated coral cells to undergo fundamental processes required for multicellular organization.

Fractionation of the three stable oxygen isotopes by oxygen-producing and oxygen-consuming reactions in photosynthetic organisms.

The triple isotope composition (delta17O and delta18O) of dissolved O2 in the ocean and in ice cores was recently used to assess the primary productivity over broad spatial and temporal scales. However, assessment of the productivity with the aid of this method must rely on accurate measurements of the 17O/16O versus 18O/16O relationship in each of the main oxygen-producing and -consuming reactions. Data obtained here showed that cleavage of water in photosystem II did not fractionate oxygen isotopes; the delta18O and delta17O of the O2 evolved were essentially identical to those of the substrate water. The fractionation slopes for the oxygenase reaction of Rubisco and respiration were identical (0.518 +/- 0.001) and that of glycolate oxidation was 0.503 +/- 0.002. There was a considerable difference in the slopes of O2 photoreduction (the Mehler reaction) in the cyanobacterium Synechocystis sp. strain PCC 6803 (0.497 +/- 0.004) and that of pea (Pisum sativum) thylakoids (0.526 +/- 0.001). These values provided clear and independent evidence that the mechanism of O2 photoreduction differs between higher plants and cyanobacteria. We used our method to assess the magnitude of O2 photoreduction in cyanobacterial cells maintained under conditions where photorespiration was negligible. It was found that electron flow to O2 can be as high as 40% that leaving photosystem II, whereas respiratory activity in the light is only 6%. The implications of our findings to the evaluation of specific O2-producing or -consuming reactions, in vivo, are discussed.

Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria.

O(2) photoreduction by photosynthetic electron transfer, the Mehler reaction, was observed in all groups of oxygenic photosynthetic organisms, but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O(2) directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O(2) photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O(2) uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P(700) was severely depressed in mutants Deltaflv1 and Deltaflv3 but recovered after light activation of CO(2) fixation, which gives P(700) an additional electron acceptor. Inhibition of CO(2) fixation prevented recovery but scarcely affected P(700) oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O(2) photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O(2).