Osmotic pressure in a bacterial swarm.

Using Escherichia coli as a model organism, we studied how water is recruited by a bacterial swarm. A previous analysis of trajectories of small air bubbles revealed a stream of fluid flowing in a clockwise direction ahead of the swarm. A companion study suggested that water moves out of the agar into the swarm in a narrow region centered ∼ 30 µm from the leading edge of the swarm and then back into the agar (at a smaller rate) in a region centered ∼ 120 µm back from the leading edge. Presumably, these flows are driven by changes in osmolarity. Here, we utilized green/red fluorescent liposomes as reporters of osmolarity to verify this hypothesis. The stream of fluid that flows in front of the swarm contains osmolytes. Two distinct regions are observed inside the swarm near its leading edge: an outer high-osmolarity band (∼ 30 mOsm higher than the agar baseline) and an inner low-osmolarity band (isotonic or slightly hypotonic to the agar baseline). This profile supports the fluid-flow model derived from the drift of air bubbles and provides new (to our knowledge) insights into water maintenance in bacterial swarms. High osmotic pressure at the leading edge of the swarm extracts water from the underlying agar and promotes motility. The osmolyte is of high molecular weight and probably is lipopolysaccharide.

Three mutations in Escherichia coli that generate transformable functional flagella.

Hydrodynamics predicts that swimming bacteria generate a propulsion force when a helical flagellum rotates because rotating helices necessarily translate at a low Reynolds number. It is generally believed that the flagella of motile bacteria are semirigid helices with a fixed pitch determined by hydrodynamic principles. Here, we report the characterization of three mutations in laboratory strains of Escherichia coli that produce different steady-state flagella without losing cell motility. E. coli flagella rotate counterclockwise during forward swimming, and the normal form of the flagella is a left-handed helix. A single amino acid exchange A45G and a double mutation of A48S and S110A change the resting flagella to right-handed helices. The stationary flagella of the triple mutant were often straight or slightly curved at neutral pH. Deprotonation facilitates the helix formation of it. The helical and curved flagella can be transformed to the normal form by torsion upon rotation and thus propel the cell. These mutations arose in the long-term laboratory cultivation. However, flagella are under strong selection pressure as extracellular appendages, and similar transformable flagella would be common in natural environments.

Coevolution of aah: a dps-like gene with the host bacterium revealed by comparative genomic analysis.

A protein named AAH was isolated from the bacterium Microbacterium arborescens SE14, a gut commensal of the lepidopteran larvae. It showed not only a high sequence similarity to Dps-like proteins (DNA-binding proteins from starved cell) but also reversible hydrolase activity. A comparative genomic analysis was performed to gain more insights into its evolution. The GC profile of the aah gene indicated that it was evolved from a low GC ancestor. Its stop codon usage was also different from the general pattern of Actinobacterial genomes. The phylogeny of dps-like proteins showed strong correlation with the phylogeny of host bacteria. A conserved genomic synteny was identified in some taxonomically related Actinobacteria, suggesting that the ancestor genes had incorporated into the genome before the divergence of Micrococcineae from other families. The aah gene had evolved new function but still retained the typical dodecameric structure.

Wood identification with PCR targeting noncoding chloroplast DNA.

Wood identification is extremely important in the modern forest industry. It also has significant applications in forensics, as well as in archeology and ecological research. In this study, five universal primer pairs amplifying chloroplast noncoding sequences of 300-1,200 bp were designed. Sequencing these amplicons in combination can lead to reliable identification of logs and wood products to cultivar, ecotype, or even the falling population. These primer pairs work on both gymnosperms and angiosperm trees. They also are potentially applicable to accurately identify shrubs and herbaceous species. In addition, a wood DNA purification method is proposed in which N-phenacylthiazolium bromide (PTB) is used to increase the quality and quantity of extracted DNA. This method was first validated using air-dried timber disks from three different tree species that were felled 4 years ago. The sapwood and outer heartwood provided the best locations for DNA extraction. The method was also successfully applied to extract DNA from the recalcitrant processed white oak wood, randomly selected staves of wine barrels. The single nucleotide polymorphism detected on the oak DNA sequences showed correlation to their geographical origins.

Tsr-GFP accumulates linearly with time at cell poles, and can be used to differentiate 'old' versus 'new' poles, in Escherichia coli.

SUMMARY: In Escherichia coli, the chemotaxis receptor protein Tsr localizes abundantly to cell poles. The current study, utilizing a Tsr-GFP fusion protein and time-lapse fluorescence microscopy of individual cell lineages, demonstrates that Tsr accumulates approximately linearly with time at the cell poles and that, in consequence, more Tsr is present at the old pole of each cell than at its newborn pole. The rate of pole-localized Tsr accumulation is large enough that old and new poles can always be reliably distinguished, even for cells whose old poles have had only one generation to accumulate signal. Correspondingly, Tsr-GFP can be reliably used to assign new and old poles to any cell without use of information regarding pole heritage, thus providing a useful tool to analyse cells whose prior history is not available. The absolute level of Tsr-GFP at the old pole of a cell also provides a rough estimate of pole (and thus cell) age.

Cloning of prokaryotic genes by a universal degenerate primer PCR.

A PCR approach was developed using a hexameric degenerate primer, which reflects the Shine-Dalgarno sequence of prokaryotic transcripts, hitherto named SD-PCR. In standard PCR reactions, the sizes and melting temperatures of the two primers are usually designed to be as equal as possible, while SD-PCR uses a single long gene-specific primer pairing with a much-shorter universal degenerate primer. This approach can be used in PCR walking to clone either the upstream or the downstream region of a known sequence. We have successfully applied the method to template DNAs of different GC contents as well as complex mixtures composed of highly contaminating DNA(s).

Bacterial motion in narrow capillaries.

Motile bacteria often have to pass through small tortuous pores in soil or tissue of higher organisms. However, their motion in this prevalent type of niche is not fully understood. Here, we modeled it with narrow glass capillaries and identified a critical radius (Rc) for bacterial motion. Near the surface of capillaries narrower than that, the swimming trajectories are helices. In larger capillaries, they swim in distorted circles. Under non-slip condition, the peritrichous Escherichia coli swam in left-handed helices with an Rc of ~10 µm near glass surface. However, slipping could occur in the fast monotrichous Pseudomonas fluorescens, when a speed threshold was exceeded, and thus both left-handed and right-handed helices were executed in glass capillaries. In the natural non-cylindrical pores, the near-surface trajectories would be spirals and twisted loops. Engaging in such motions reduces the bacterial migration rate. With a given pore size, the run length and the tumbling angle of the bacterium determine the probability and duration of their near-surface motion. Shear flow and chemotaxis potentially enhance it. Based on this observation, the puzzling previous observations on bacterial migration in porous environments can be interpreted.

Confinement-dependent localization of diffusing aggregates in cellular geometries.

Confinement has a strong influence on diffusing nano-sized clusters. In particular, biomolecular aggregates within the shell-like confining space of a bacterial cell have been shown to display a variety of localization patterns, from being midcell to the poles. How does the confining space determine where the aggregate will localize? Here, using Monte Carlo simulations we have calculated the equilibrium spatial distribution of fixed-sized clusters diffusing in spherocylindrical shells. We find that localization to the poles depends strongly on shell thickness and the size of the cluster. Compared to being at midcell, polar clusters can be more bent and hence have higher energy, but they also can have a greater number of defects and hence have more entropy. Under certain conditions this can lead to polar clusters having a lower free energy than at midcell, favoring localization to the poles. Our findings suggest possible localization selection mechanisms within shell-like geometries that can arise purely from cluster confinement.

Swimming behavior of the monotrichous bacterium Pseudomonas fluorescens SBW25.

Motility is an important trait for some bacteria living in nature and the analyses of it can provide important information on bacterial ecology. While the swimming behavior of peritrichous bacteria such as Escherichia coli has been extensively studied, the monotrichous bacteria such as the soil inhabiting and plant growth promoting bacterium Pseudmonas fluorescens is not very well characterized. Unlike E. coli that is propelled by a left-handed flagella bundle, P. fluorescens SBW25 swims several times faster by rotating a right-handed flagellum. Its swimming pattern is the most sophisticated known so far: it swims forward (run) and backward (backup); it can swiftly 'turn' the run directions or 'reorient' at run-backup transitions; it can 'flip' the cell body continuously or 'hover' in the milieu without translocation. The bacteria swam in circles near flat surfaces with reduced velocity and increased turn frequency. The viscous drag load due to wall effect potentially accounts for the circular motion and velocity change, but not the turn frequency. The flagellation and swimming behavior of P. fluorescens SBW25 show some similarity to Caulobacter, a fresh-water inhabitant, while the complex swimming pattern might be an adaptation to the geometrically restricted rhizo- and phyllospheres.

Cell orientation of swimming bacteria: from theoretical simulation to experimental evaluation.

The motion of small bacteria consists of two phases: relatively long runs alternate with intermittent stops, back-ups, or tumbles, depending on the species. In polar monotrichous bacteria, the flagellum is anchored at the cell pole inherited from the parent generation (old pole) and is surrounded by a chemoreceptor cluster. During forward swimming, the leading pole is always the pole recently formed in cell division (new pole). The flagella of the peritrichous bacterium Escherichia coli often form a bundle behind the old pole. Its cell orientation and receptor positioning during runs generally mimic that of monotrichous bacteria. When encountering a solid surface, peritrichous bacteria exhibit a circular motion with the leading pole dipping downward. Some polar monotrichous bacteria also perform circular motion near solid boundaries, but during back-ups. In this case, the leading pole points upward. Very little is known about behavior near milieu-air interfaces. Biophysical simulations have revealed some of the mechanisms underlying these phenomena, but leave many questions unanswered. Combining biophysics with molecular techniques will certainly advance our understanding of bacterial locomotion.

The nonribosomal peptide and polyketide synthetic gene clusters in two strains of entomopathogenic fungi in Cordyceps.

Species of Cordyceps Fr. are entomopathogenic fungi that parasitize the larvae or pupae of lepidopteran insects. The secondary metabolites, nonribosomal peptides and polyketides are well-known mediators of pathogenesis. The biosynthetic gene clusters of these compounds in two fungal strains (1630 and DSM 1153) formerly known as Cordyceps militaris were screened using polymerase chain reaction with degenerate primers. Two nonribosomal peptide synthetase genes, one polyketide synthetase gene and one hybrid gene cluster were identified, and certain characteristics of the structures of their potential products were predicted. All four genes were actively expressed under laboratory conditions but at markedly different levels. The gene clusters from the two fungal strains were structurally and functionally unrelated, suggesting different evolutionary origins and physiological functions. Phylogenetic and biochemical analyses confirmed that the two fungal strains are not conspecific as currently assigned.

Gender-specific bacterial composition of black flies (Diptera: Simuliidae).

Although hematophagous black flies are well-known socioeconomic pests and vectors of disease agents, their associated bacteria are poorly known. A systematic analysis of the bacterial community associated with freshly emerged adult black flies of four North American species, using cultivation-independent molecular techniques, revealed 75 nonsingleton bacterial phylotypes. Although 17 cosmopolitan phylotypes were shared among host species, each fly species had a distinct bacterial profile. The bacterial composition, however, did not correlate strongly with the host phylogeny but differed between male and female flies of the same species from the same habitat, demonstrating that a group of insects have a gender-dependent bacterial community. In general, female flies harbor a less diverse bacterial community than do males. The anatomical locations of selected bacteria were revealed using fluorescence in situ hybridization. Understanding the physiological function of the associated bacterial community could provide clues for developing novel pest-management strategies.

Comparative evaluation of the gut microbiota associated with the below- and above-ground life stages (larvae and beetles) of the forest cockchafer, Melolontha hippocastani.

A comparison of the diversity of bacterial communities in the larval midgut and adult gut of the European forest cockchafer (Melolontha hippocastani) was carried out using approaches that were both dependent on and independent of cultivation. Clone libraries of the 16S rRNA gene revealed 150 operational taxonomic units (OTUs) that belong to 11 taxonomical classes and two other groups that could be classified only to the phylum level. The most abundant classes were ß, δ and γ-proteobacteria, Clostridia, Bacilli, Erysipelotrichi and Sphingobacteria. Although the insect's gut is emptied in the prepupal stage and the beetle undergoes a long diapause period, a subset of eight taxonomic classes from the aforementioned eleven were found to be common in the guts of diapausing adults and the larval midguts (L2, L3). Moreover, several bacterial phylotypes belonging to these common bacterial classes were found to be shared by the larval midgut and the adult gut. Despite this, the adult gut bacterial community represented a subset of that found in the larvae midgut. Consequently, the midgut of the larval instars contains a more diverse bacterial community compared to the adult gut. On the other hand, after the bacteria present in the larvae were cultivated, eight bacterial species were isolated. Moreover, we found evidence of the active role of some of the bacterial species isolated in food digestion, namely, the presence of amylase and xylanolytic properties. Finally, fluorescence in situ hybridization allowed us to confirm the presence of selected species in the insect gut and through this, their ecological niche as well as the metagenomic results. The results presented here elucidated the heterogeneity of aerobic and facultative bacteria in the gut of a holometabolous insect species having two different feeding habits.

Vital dye reaction and granule localization in periplasm of Escherichia coli.

BACKGROUND: Tetrazolium salts are widely used in biology as indicators of metabolic activity - hence termed vital dyes - but their reduction site is still debated despite decades of intensive research. The prototype, 2,3,5- triphenyl tetrazolium chloride, which was first synthesized a century ago, often generates a single formazan granule at the old pole of Escherichia coli cells after reduction. So far, no explanation for their pole localization has been proposed. METHOD/PRINCIPAL FINDINGS: Here we provide evidence that the granules form in the periplasm of bacterial cells. A source of reducing power is deduced to be thiol groups destined to become disulfides, since deletion of dsbA, coding for thiol-oxidase, enhances the formation of reduced formazan. However, pervasive reduction did not result in a random distribution of formazan aggregates. In filamentous cells, large granules appear at regular intervals of about four normal cell-lengths, consistent with a diffusion-to-capture model. Computer simulations of a minimal biophysical model showed that the pole localization of granules is a spontaneous process, i.e. small granules in a normal size bacterium have lower energy at the poles. This biased their diffusion to the poles. They kept growing there and eventually became fixed. CONCLUSIONS: We observed that formazan granules formed in the periplasm after reduction of tetrazolium, which calls for re-evaluation of previous studies using cell-free systems that liberate inaccessible intracellular reductant and potentially generate artifacts. The localization of formazan granules in E. coli cells can now be understood. In living bacteria, the seeds formed at or migrated to the new pole would become visible only when that new pole already became an old pole, because of the relatively slow growth rate of granules relative to cell division.

Complexity and variability of gut commensal microbiota in polyphagous lepidopteran larvae.

BACKGROUND: The gut of most insects harbours nonpathogenic microorganisms. Recent work suggests that gut microbiota not only provide nutrients, but also involve in the development and maintenance of the host immune system. However, the complexity, dynamics and types of interactions between the insect hosts and their gut microbiota are far from being well understood. METHODS/PRINCIPAL FINDINGS: To determine the composition of the gut microbiota of two lepidopteran pests, Spodoptera littoralis and Helicoverpa armigera, we applied cultivation-independent techniques based on 16S rRNA gene sequencing and microarray. The two insect species were very similar regarding high abundant bacterial families. Different bacteria colonize different niches within the gut. A core community, consisting of Enterococci, Lactobacilli, Clostridia, etc. was revealed in the insect larvae. These bacteria are constantly present in the digestion tract at relatively high frequency despite that developmental stage and diet had a great impact on shaping the bacterial communities. Some low-abundant species might become dominant upon loading external disturbances; the core community, however, did not change significantly. Clearly the insect gut selects for particular bacterial phylotypes. CONCLUSIONS: Because of their importance as agricultural pests, phytophagous Lepidopterans are widely used as experimental models in ecological and physiological studies. Our results demonstrated that a core microbial community exists in the insect gut, which may contribute to the host physiology. Host physiology and food, nevertheless, significantly influence some fringe bacterial species in the gut. The gut microbiota might also serve as a reservoir of microorganisms for ever-changing environments. Understanding these interactions might pave the way for developing novel pest control strategies.

Crystallization of α- and ß-carotene in the foregut of Spodoptera larvae feeding on a toxic food plant.

In the animal kingdom, carotenoids are usually absorbed from dietary sources and transported to target tissues. Despite their general importance, the uptake mechanism is still poorly understood. Here we report the "red crop" phenomenon, an accumulation of α- and ß-carotene in crystalline inclusions in the enlarged foregut of the polyphagous Spodoptera larvae feeding on some potentially toxic plant leaves. The carotene crystals give the insect foregut a distinctive orange-red color. The crystals are embedded in a homogenous lawn of the bacterium Enterococcus casseliflavus, but the carotene seems to be selectively taken from the food plant. Caterpillars which fail to develop these carotene crystals exhibit a high mortality or fail to develop to adulthood. The crystallization of carotene and the enlargement of the foregut thus appears to manifest a multiple-step physiological adaptation of the insects to toxic food plants.

The asymmetric flagellar distribution and motility of Escherichia coli.

Rod-shaped bacteria such as Escherichia coli divide by binary fission. They inherit an old pole from the parent cell. The new pole is recently derived from the septum. Because the chemoreceptor accumulates linearly with time on the cell pole, the old pole carries more receptors than does the new pole. Here, further evidence is provided that the old pole appears more frequently at the rear when bacteria swim. This phenomenon had been observed, yet not extensively explored in the literature. The biased swimming orientation is the consequence of the asymmetric distribution of flagella over the cell surface. On about 75% of cells, there are more flagella on the old-pole half of the cell than on the new-pole half, regardless of growth conditions. Most flagella are lateral, and few were found on the cell pole per se. The asymmetric flagellar distribution makes cells more efficient in chemotaxis. Both swimming orientation and receptor localization are components of chemotaxis, by which bacteria follow environmental stimuli. If unipolarly flagellated cells, such as the swarmer cells of Caulobacter crescentus, are regarded as 100% polar with respect to chemotaxis, E. coli is about 75%. The difference is quantitative. The peritrichous flagellation might enhance the motility and chemotaxis in the viscous environment of enteric bacteria.

Methyl group migration during the fragmentation of singly charged ions of trimethyllysine-containing peptides: precaution of using MS/MS of singly charged ions for interrogating peptide methylation.

Core histones are susceptible to a range of post-translational modifications (PTMs), including acetylation, phosphorylation, methylation, and ubiquitination, which play important roles in the epigenetic control of gene expression. Here, we observed an unusual discrepancy between MALDI-MS/MS and ESI-MS/MS on the methylation of trimethyllysine-containing peptides with residues 9-17 from human histone H3 and residues 73-83 from yeast histone H3. It turned out that the discrepancy could be attributed to an unusual methyl group migration from the side chain of trimethyllysine to the C-terminal arginine residue during peptide fragmentation, and this methyl group transfer only occurred for singly charged ions, but not for doubly charged ions. The methyl group transfer argument received its support from the results on the studies of the fragmentation of the ESI- or MALDI-produced singly charged ions of several synthetic trimethyllysine-bearing peptides. The results presented in this study highlighted that caution should be exerted while MS/MS of singly charged ions is employed to interrogate the PTMs of trimethyllysine-containing peptides.

A novel Dps-type protein from insect gut bacteria catalyses hydrolysis and synthesis of N-acyl amino acids.

A novel type of a microbial N-acyl amino acid hydrolase (AAH) from insect gut bacteria was purified, cloned and functionally characterized. The enzyme was obtained from Microbacterium arborescens SE14 isolated from the foregut of larvae of the generalist herbivore Spodoptera exigua. The substrates of AAH are N-acyl-glutamines previously reported to elicit plant defence reactions after introduction into the leaf during feeding. The isolated AAH catalyses the hydrolysis of the amide bond (K(m) = 36 micromol l(-1)) and, less efficient, the formation (K(m) = 3 mmol l(-1)) of the elicitor active N-acyl amino acids. The AAH from M. arborescens SE14 shows no homology to known fatty acyl amidases (EC 3.5.1.4) but belongs to the family of Dps proteins (DNA-binding protein from starved cell). In line with other DPS proteins AAH is a homododecamer (monomer 17 181 Da) and contains iron atoms (c. 1-16 iron atoms per subunit). Unlike genuine DPS proteins the enzyme does not significantly bind DNA. Amino acid hydrolase is the first member of the DPS family that catalyses the cleavage or formation of amide bonds. The participation of a microbial enzyme in the homeostasis of N-acyl-glutamines in the insect gut adds further complexity to the interaction between plants and their herbivores.