The effects of Bacillus subtilis on Caenorhabditis elegans fitness after heat stress

Microbes can provide their hosts with protection from biotic and abiotic factors. While many studies have examined how certain bacteria can increase host lifespan, fewer studies have examined how host reproduction can be altered. The nematode Caenorhabditis elegans has been a particularly useful model system to examine how bacteria affect the fitness of their hosts under different contexts. Here, we examine how the bacterium Bacillus subtilis, compared to the standard C. elegans lab diet, Escherichia coli, affects C. elegans survival and reproduction after experiencing a period of intense heat stress. We find that under standard conditions, nematodes reared on B. subtilis produce fewer offspring than when reared on E. coli.However, despite greater mortality rates on B. subtilis after heat shock, young adult nematodes produced more offspring after heat shock when fed B. subtilis compared to E. coli. Because offspring production is necessary for host population growth and evolution, the reproductive advantage conferred by B. subtilis supersedes the survival advantage of E. coli. Furthermore, we found that nematodes must be reared on B. subtilis (particularly at the early stages of development) and not merely be exposed to the bacterium during heat shock, to obtain the reproductive benefits provided by B. subtilis. Taken together, our findings lend insight into the importance of environmental context and interaction timing in shaping the protective benefits conferred by a microbe toward its host.     

Chemotaxis toward amino acids inBacillus subtilis

Quantitative measurement of positive chemotaxis inBacillus subtilis was performed by means of adaption of the procedure used in studies withEscherichia coli. The motility ofB. subtilis was optimal in the presence of an exogeneous energy source and a nonionic detergent,e. g. Tween 80 or Brij-36. B. subtilis is chemotactic toward the commonly occurringL-amino acids except arginine, lysine, aspartate and glutamate. No chemotactic response was observed towardD-amino acids. Threshold, optimal response and peak concentration were determined. Chemotaxis toward glutamine was optimal at pH 6-7 and a temperature of 32°C. The maximum response toward a particular attractant was presumably influenced by the aerotactic behavior ofB. subtilis.     

The effect of amino acids on the motile behavior of Bacillus subtilis

Constant levels of amino acids enhanced the velocity of Bacillus subtilis 60015 cells about 2-fold and stimulated the response in motility assays. The stimulation of velocity did not occur via the receptors for chemotaxis. Cysteine and methionine, general inhibitors of chemotaxis, both completely inhibited the smooth response in a temporal gradient of attractant. After methionine starvation B. subtilis 60015 showed no measurable response in a temporal gradient of attractant, this in contrast to the effect observed with some other bacteria. Addition of methionine to starved cells restored the response toward attractant. Revertants of B. subtilis 60015 for methionine requirement could not be starved and showed a normal behavior toward temporal gradients of attractant.Abbreviation O.D.₆₀₀ optical density measured at 600 nm     

Chemotaxis of Bradyrhizobium japonicum to soybean exudates.

The chemotactic response of Bradyrhizobium japonicum toward soybean seed and root exudates was examined. Assays using various isoflavones and fractionated exudate indicated that isoflavones are not the principal attractants in exudates. Likewise, induction of nod genes with isoflavones or seed exudate before assay did not enhance chemotaxis. Screening of numerous compounds revealed that only dicarboxylic acids and the amino acids glutamate and aspartate were strong attractants. The presence of glutamate, aspartate, and dicarboxylic acids in appreciable concentrations in soybean seed and root exudates indicates that these compounds likely represent natural chemoattractants for B. japonicum.     

Chemotaxis of Bradyrhizobium japonicum to soybean exudates.

The chemotactic response of Bradyrhizobium japonicum toward soybean seed and root exudates was examined. Assays using various isoflavones and fractionated exudate indicated that isoflavones are not the principal attractants in exudates. Likewise, induction of nod genes with isoflavones or seed exudate before assay did not enhance chemotaxis. Screening of numerous compounds revealed that only dicarboxylic acids and the amino acids glutamate and aspartate were strong attractants. The presence of glutamate, aspartate, and dicarboxylic acids in appreciable concentrations in soybean seed and root exudates indicates that these compounds likely represent natural chemoattractants for B. japonicum.     

Effect of temperature on Pseudomonas fluorescens chemotaxis.

The effects of temperature and attractants on chemotaxis in psychrotrophic Pseudomonas fluorescens were examined using the Adler capillary assay technique. Several organic acids, amino acids, and uronic acids were shown to be attractants, whereas glucose and its oxidation products, gluconate and 2-ketogluconate, elicited no detectable response. Chemotaxis toward many attractants was dependent on prior growth of the microorganism with these compounds. However, the organic acids, malate and succinate, caused strong chemotactic responses regardless of the carbon source used for growth of the bacteria. The temperature at which the cells were grown (30 or 5 degrees C) had no significant detectable effect on chemotaxis to the above attractants. The temperature at which the cells were assayed appeared to affect the rate but the extent of the chemotactic response, nor the concentration response curves. The ratios of the rate of accumulation of cells to the attractant malate were approximately 2, 4, and 1 at 30, 17, and 5 degrees C, respectively. Strong chemotactic responses were observed with cells assayed at temperatures approaching 0 degree C and appeared to be functional over a broad temperature range of 3 to 35 degrees C.     

Chemotaxis and transport of amino acids in Allomyces arbuscula

Among a number of amino acids tested, l-lysine and l-arginine are the principal attractants in the chemotaxis of the zygotes of Allomyces arbuscula. The reaction can be stimulated to a greater or lesser extent by a number of compounds chemically related to l-leucine. No relationship between transport of attracting amino acids and their effect on chemotaxis has been found.     

Regulation of Pseudomonas aeruginosa chemotaxis by the nitrogen source.

The regulation of amino acid chemotaxis by nitrogen was investigated in the gram-negative bacterium Pseudomonas aeruginosa. The quantitative capillary tube technique was used to measure chemotactic responses of bacteria to spatial gradients of amino acids and other attractants. Chemotaxis toward serine, arginine, and alpha-aminoisobutyrate was sharply dependent on the form in which nitrogen was presented to the bacteria. Bacteria grown on mineral salts-succinate with potassium nitrate gave responses to amino acids that were 2 to 3 times those of cells grown on ammonium sulfate and 10 to 20 times those of cells grown in mineral salts-succinate with Casamino Acids as the nitrogen source. A combination of ammonium sulfate and glutamate was as effective as Casamino Acids in depressing serine taxis. The threshold concentration for alpha-aminoisobutyrate taxis was consistently lower in nitrate-grown bacteria than in ammonia-grown bacteria. Responsiveness to sodium succinate, however, was not subject to regulation by nitrogen, and glucose chemotaxis was inhibited, rather than enhanced, in nitrate-grown bacteria. These results indicate that chemotaxis of P. aeruginosa toward amino acids is subject to regulation by nitrogen and that this regulation probably is expressed at the level of the chemoreceptors or transducers.     

Chemotactic Behavior of Pathogenic and Nonpathogenic Leptospira Species

We have developed a capillary tube assay in combination with real-time PCR to quantitate the number of chemoattracted Leptospira cells. We identified Tween 80, glucose, sucrose, and pyruvate as attractants for Leptospira cells; amino acids and vitamin B₁₂ were found to be nonchemotactic or weakly chemotactic. This assay has the general applicability to further our understanding of leptospiral chemotaxis.     

Three unrelated chemoreceptors provide Pectobacterium atrosepticum with a broad-spectrum amino acid sensing capability

Amino acids are important nutrients and also serve as signals for diverse signal transduction pathways. Bacteria use chemoreceptors to recognize amino acid attractants and to navigate their gradients. In Escherichia coli two likely paralogous chemoreceptors Tsr and Tar detect 9 amino acids, whereas in Pseudomonas aeruginosa the paralogous chemoreceptors PctA, PctB and PctC detect 18 amino acids. Here, we show that the phytobacterium Pectobacterium atrosepticum uses the three non-homologous chemoreceptors PacA, PacB and PacC to detect 19 proteinogenic and several non-proteinogenic amino acids. PacB recognizes 18 proteinogenic amino acids as well as 8 non-proteinogenic amino acids. PacB has a ligand preference for the three branched chain amino acids L-leucine, L-valine and L-isoleucine. PacA detects L-proline next to several quaternary amines. The third chemoreceptor, PacC, is an ortholog of E. coli Tsr and the only one of the 36 P. atrosepticum chemoreceptors that is encoded in the cluster of chemosensory pathway genes. Surprisingly, in contrast to Tsr, which primarily senses serine, PacC recognizes aspartate as the major chemoeffector but not serine. Our results demonstrate that bacteria use various strategies to sense a wide range of amino acids and that it takes more than one chemoreceptor to achieve this goal.     

Imprecision of Adaptation in Escherichia coli Chemotaxis

Adaptability is an essential property of many sensory systems, enabling maintenance of a sensitive response over a range of background stimulus levels. In bacterial chemotaxis, adaptation to the preset level of pathway activity is achieved through an integral feedback mechanism based on activity-dependent methylation of chemoreceptors. It has been argued that this architecture ensures precise and robust adaptation regardless of the ambient ligand concentration, making perfect adaptation a celebrated property of the chemotaxis system. However, possible deviations from such ideal adaptive behavior and its consequences for chemotaxis have not been explored in detail. Here we show that the chemotaxis pathway in Escherichia coli shows increasingly imprecise adaptation to higher concentrations of attractants, with a clear correlation between the time of adaptation to a step-like stimulus and the extent of imprecision. Our analysis suggests that this imprecision results from a gradual saturation of receptor methylation sites at high levels of stimulation, which prevents full recovery of the pathway activity by violating the conditions required for precise adaptation. We further use computer simulations to show that limited imprecision of adaptation has little effect on the rate of chemotactic drift of a bacterial population in gradients, but hinders precise accumulation at the peak of the gradient. Finally, we show that for two major chemoeffectors, serine and cysteine, failure of adaptation at concentrations above 1 mM might prevent bacteria from accumulating at toxic concentrations of these amino acids.     

Chemotaxis by Naegleria fowleri for Bacteria1

Naegleria fowleri amebae demonstrated a chemotactic and chemokinetic response toward live cells and extracts of Escherichia coli and other bacterial species when experiments were performed using a blind-well chemotaxis chamber. The peptide N-formyl-methionyl-leucyl-phenylalanine acted as a chemokinetic rather than a chemotactic factor for N. fowleri amebae. Competition experiments in which nerve cell extracts or bacteria were placed on either side of the filter in chemotaxis chambers resulted in increased movement towards bacteria. A scanning electron microscopy study of the interaction of N. fowleri with different bacterial species confirmed that when the amebae were near ingestible bacteria they moved toward the bacteria by pseudopod formation. Naegleria fowleri appeared to respond to bacteria by three interrelated but distinct processes: (a) chemokinesis, (b) chemotaxis, and (c) formation of food cups.     

Diversity in Chemotaxis Mechanisms among the Bacteria and Archaea

The study of chemotaxis describes the cellular processes that control the movement of organisms toward favorable environments. In bacteria and archaea, motility is controlled by a two-component system involving a histidine kinase that senses the environment and a response regulator, a very common type of signal transduction in prokaryotes. Most insights into the processes involved have come from studies of Escherichia coli over the last three decades. However, in the last 10 years, with the sequencing of many prokaryotic genomes, it has become clear that E. coli represents a streamlined example of bacterial chemotaxis. While general features of excitation remain conserved among bacteria and archaea, specific features, such as adaptational processes and hydrolysis of the intracellular signal CheY-P, are quite diverse. The Bacillus subtilis chemotaxis system is considerably more complex and appears to be similar to the one that existed when the bacteria and archaea separated during evolution, so that understanding this mechanism should provide insight into the variety of mechanisms used today by the broad sweep of chemotactic bacteria and archaea. However, processes even beyond those used in E. coli and B. subtilis have been discovered in other organisms. This review emphasizes those used by B. subtilis and these other organisms but also gives an account of the mechanism in E. coli.     

Characterization of methyl-accepting chemotaxis proteins (MCPs) for amino acids in plant-growth-promoting rhizobacterium Pseudomonas protegens CHA0 and enhancement of amino acid chemotaxis by MCP genes overexpression

ABSTRACT

Pseudomonas protegens CHA0, known as plant-growth-promoting rhizobacterium, showed positive chemotactic responses toward proteinaceous L-amino acids. Genomic analysis revealed that P. protegens CHA0 possesses four putative chemoreceptors for amino acids (designated CtaA, CtaB, CtaC, and CtaD, respectively). Pseudomonas aeruginosa PCT2, a mutant defective in chemotaxis to amino acids, harboring a plasmid containing each of ctaA, ctaB, ctaC, and ctaD showed chemotactic responses to 20, 4, 4, and 11 types of amino acids, respectively. To enhance chemotaxis toward amino acids, we introduced the plasmids containing ctaA, ctaB, ctaC, or ctaD into P. protegens CHA0. By overexpression of the genes, we succeeded in enhancing chemotaxis toward more than half of the tested ligands. However, unexpectedly, the P. protegens CHA0 transformants showed unchanged or decreased responses to some amino acids when compared to wild-type CHA0. We speculate that alternation of expression of a chemoreceptor may affect the abundance of other chemoreceptors.     

Bacillus subtilis spore protein SpoVAC functions as a mechanosensitive channel

A critical event during spore germination is the release of Ca‐DPA (calcium in complex with dipicolinic acid). The mechanism of release of Ca‐DPA through the inner membrane of the spore is not clear, but proteins encoded by the Bacillus subtilis spoVA operon are involved in the process. We cloned and expressed the spoVAC gene in Escherichia coli and characterized the SpoVAC protein. We show that SpoVAC protects E. coli against osmotic downshift, suggesting that it might act as a mechanosensitive channel. Purified SpoVAC was reconstituted in unilamellar lipid vesicles to determine the gating mechanism and pore properties of the protein. By means of a fluorescence‐dequenching assay, we show that SpoVAC is activated upon insertion into the membrane of the amphiphiles lysoPC and dodecylamine. Patch clamp experiments on E. coli giant spheroplast as well as giant unilamellar vesicles (GUVs) containing SpoVAC show that the protein forms transient pores with main conductance values of about 0.15 and 0.1 nS respectively. Overall, our data indicate that SpoVAC acts as a mechanosensitive channel and has properties that would allow the release of Ca‐DPA and amino acids during germination of the spore.     

Chemotaxis toward carbohydrates and amino acids in Physarum polycephalum

A method is described for assaying chemotaxis in the acellular slime mold Physarum polycephalum. It consists of measuring the amount of plasmodium that moves on a strip of nitrocellulose membrane filter Millipore in response to a gradient of an attractant. Time course of chemotactic response of the slime mold is described. Different factors that affect chemotaxis in the slime mold such as: culture care and stage of growth of microplasmodia, substratum used for cell movement, nature of the gradient, effect of salts, pH and temperature are described. From concentration-response curves for different attractants several parameters of the chemotactic effect, such as threshold concentration, half maximal concentration, and maximal effective concentration can be determined. As a group, sugars are more effective chemotactic agents than amino acids. Glucose and galactose, which support the growth of the slime mold, are shown to have high positive chemotactic effect. 3-O-Methyl- -glucose and 2-deoxy- -glucose are two sugars that do not support growth but are very effective attractants. Conversely, fructose which supports slime mold growth is at best a weak attractant. The results support the view that the chemotactic effects of different sugars are not dependent on their growth-supporting value.     

Chemical characterization of root exudates from rice (Oryza sativa) and their effects on the chemotactic response of endophytic bacteria

Root exudates represent an important source of nutrients for microorganisms in the rhizosphere and seem to participate in early colonization inducing chemotactic responses of rhizospheric bacteria. We characterized the root exudates collected from rice plantlets cultured under hydroponic conditions and assessed their effects on the chemotaxis of two strains of endophytic bacteria, Corynebacterium flavescens and Bacillus pumilus, collected from the rice rhizosphere. We compared these chemotactic effects on endophytic bacteria with those on two strains of plant-growth-promoting bacteria, Azospirillum brasilense (isolated from the corn rhizosphere) and Bacillus sp. (from the rice rhizosphere). The root exudates were collected at different time intervals. The highest concentration and diversity of amino acids and carbohydrates were found during the first 2 weeks after seeding. Histidine, proline, valine, alanine, and glycine were the main amino acid residues identified during the 4 weeks of culture. The main carbohydrates identified were glucose, arabinose, mannose, galactose, and glucuronic acid. The chemotactic responses of the analyzed endophytic bacteria to root exudates were 3.9 to 5.1 times higher than those of A. brasilense and 2.2 to 2.8 times higher than Bacillus sp. Our results indicate that rice exudates may induce a higher chemotactic response for endophytic bacteria than for other bacterial strains present in the rice rhizosphere.     

A Lysine Cluster in Domain II of Bacillus subtilis PBP4a Plays a Role in the Membrane Attachment of This C1-PBP

In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K) residues, K86, K114, K119, and K265, protrude from domain II. Replacement of these amino acids with glutamine (Q) residues by site-directed mutagenesis yielded Mut4KQ PBP4a. When produced in Escherichia coli without its predicted Sec-signal peptide, wild-type (WT) PBP4a was found mainly associated with the host cytoplasmic membrane, whereas Mut4KQ PBP4a remained largely unbound. After purification, the capacities of the two proteins to bind to B. subtilis membranes were compared. The results were similar to those obtained in E. coli: in vitro, a much higher percentage of WT PBP4a than of Mut4KQ PBP4a was found to interact with B. subtilis membranes. Immunodetection of PBP4a in B. subtilis membrane extracts revealed that a processed form of this PBP (as indicated by its size) associates with the B. subtilis cytoplasmic membrane. In the absence of any amphiphilic peptide in PBP4a, the crown of positive charges on the surface of domain II is likely responsible for the cellular localization of this PBP and its attachment to the cytoplasmic membrane.     

Effect of organic amendments on bacterial multiplication in lake water

In Cayuga Lake water amended with 30 g of glucose or amino acids per ml, an added strain of Pseudomonas fluorescens and indigenous bacteria grew extensively, Pseudomonas sp. B4 and two rhizobia multiplied at a moderate extent, and introduced Escherichia coli and Klebsiella pneumoniae multiplied but to only a slight degree. The amendments did not enhance growth of Micrococcus flavus and Arthrobacter citreus, and an asporogenous strain of Bacillus subtilis decreased in numbers. The pseudomonads, rhizobia, E. coli and K. pneumoniae multiplied extensively when inoculated into sterile lake water amended with amino acids, A. citreus grew to a slight extent, but the numbers of M. flavus and B. subtilis did not change appreciably. In nonsterile lake water amended with 30 g of Trypticase soy broth per ml, the indigenous bacteria greatly increased in abundance, the pseudomonads, rhizobia, and E. coli developed to a lesser extent, the numbers of K. pneumoniae, A. citreus and M. flavus showed little increase, and B. subtilis decreased in numbers. Tests in pure culture containing 2 to 64 g of Trypticase soy broth per ml demonstrated good growth of P. fluorescens, Pseudomonas sp. B4, and the rhizobia at all concentrations; an initial decline followed by growth of E. coli, K. pneumoniae and R. leguminosarum biovar phaseoli at low concentrations; little or no growth or decline at the low levels but multiplication at the high levels by A. citreus and B. subtilis; and decline of M. flavus. It is proposed that the apparent K_s value, _max value, length of lag phase and resistance to stress can be used to predict behavior of bacteria in lake water receiving low levels of organic nutrients.     

Small stable RNA maturation and turnover in Bacillus subtilis

Stable RNA maturation is a key process in the generation of functional RNAs, and failure to correctly process these RNAs can lead to their elimination through quality control mechanisms. Studies of the maturation pathways of ribosomal RNA and transfer RNA in Bacillus subtilis showed they were radically different from Escherichia coli and led to the identification of new B. subtilis‐specific enzymes. We noticed that, despite their important roles in translation, a number of B. subtilis small stable RNAs still did not have characterised maturation pathways, notably the tmRNA, involved in ribosome rescue, and the RNase P RNA, involved in tRNA maturation. Here, we show that tmRNA is matured by RNase P and RNase Z at its 5′ and 3′ extremities, respectively, whereas the RNase P RNA is matured on its 3′ side by RNase Y. Recent evidence that several RNases are not essential in B. subtilis prompted us to revisit maturation of the scRNA, a component of the signal recognition particle involved in co‐translational insertion of specific proteins into the membrane. We show that RNase Y is also involved in 3′ processing of scRNA. Lastly, we identified some of the enzymes involved in the turnover of these three stable RNAs.