Molecular Characterization of the Nonphotosynthetic Partner Bacterium in the Consortium “Chlorochromatium aggregatum”

Phototrophic consortia represent valuable model systems for the study of signal transduction and coevolution between different bacteria. The phototrophic consortium “Chlorochromatium aggregatum” consists of a colorless central rod-shaped bacterium surrounded by about 20 green-pigmented epibionts. Although the epibiont was identified as a member of the green sulfur bacteria, and recently isolated and characterized in pure culture, the central colorless bacterium has been identified as a member of the β-Proteobacteria but so far could not be characterized further. In the present study, “C. aggregatum” was enriched chemotactically, and the 16S rRNA gene sequence of the central bacterium was elucidated. Based on the sequence information, fluorescence in situ hybridization probes targeting four different regions of the 16S rRNA were designed and shown to hybridize exclusively to cells of the central bacterium. Phylogenetic analyses of the 1,437-bp-long sequence revealed that the central bacterium of “C. aggregatum” represents a so far isolated phylogenetic lineage related to Rhodoferax spp., Polaromonas vacuolata, and Variovorax paradoxus within the family Comamonadaceae. The majority of relatives of this lineage are not yet cultured and were found in low-temperature aquatic environments or aquatic environments containing xenobiotica or hydrocarbons. In CsCl-bisbenzimidazole equilibrium density gradients, genomic DNA of the central bacterium of “Chlorochromatium aggregatum” formed a distinct band which could be detected by quantitative PCR using specific primers. Using this method, the G+C content of the central bacterium was determined to be 55.6 mol%.     

Freshwater nematodes of the Galápagos

Freshwater nematodes of the Galápagos archipelago were studied from four samples collected in 1988. Eighteen species, two of which are new to science, belonging to five orders, Enoplida, Dorylaimida, Mononchida, Monhysterida and Chromadorida, are described. Alaimus wittmeri n.sp. is characterised by having a smooth cuticle, a more posteriorly enlarged pharynx, a posteriorly situated amphidial fusus, obscure but anteriorly situated excretory pore, a mono-opisthodelphic ovary, and by the absence of sexual dimorphism in the lip region, the absence of pre-vulval uterine sac, the presence of males that have few (4–7) and weak ventromedian supplements and absence of mucro on tail tip. Monhystera floreanae n. sp. is distinguished by its females having a small body (L = 492–634 µm) with a narrow lip region, narrow posteriorly located amphidial openings, a small value for the c'-ratio (c' = 6.0–7.5), weak vaginal sphincter, by the absence of post-vaginal gland cells, absence of males and absence of crystalloid bodies. The difficulty in the use of crystalloid bodies as diagnostic character in the genus Monhystera is discussed. Two forms, a big form and a small form, which are similar except in measurements are reported for Achromadora micoletzkyi. SEM photos of ruptured cuticle of A. micoletzkyi and A. semiarmata show unique intra-cuticular structures. Comparison of these structures in the two species is made. Papilliform instead of setiform outer labial sensilla are also observed in A. micoletzkyi. The presence of the male and fine crystalloid bodies in Achromadora semiarmata, dorsal body pore in Prismatolaimus dolichurus and Prismatolaimus kenyensis, forty longitudinal cuticular ridges around the mid-body of Stomachoglossa pachyderma, crystalloid bodies in Ironus dentifurcatus are reported here for the first time. SEM photos of twelve species: Monhystera floreanae n. sp., Monhystera somereni, Alaimus wittmeri n. sp., Monhystrella hastata, Paramphidelus dolichurus, Eutobrilus annetteae, Ironus dentifurcatus, Chronogaster cameroonensis, Rhabdolaimus terrestris, Stomachoglossa pachyderma, Achromadora micoletzkyi and Achromadora semiarmata, are also presented.The richest nematode fauna was found in El Junco (San Cristobal), the only permanent freshwater lake on the archipelago. The nine species recorded from the lake are probably only a fraction of the total nematode fauna. The very recent man made reservoir La Toma has a relatively high diversity, due to its vicinity to El Junco. It is argued that the freshwater nematodes from San Cristobal probably arrived there through passive transport by birds.The freshwater bodies encountered on Floreana are very different from those on San Cristobal and so is their nematode fauna. Part of the species found on these islands may have been transported by land birds, others may have invaded the freshwater from terrestrial habitats.Apart from the two new species, the other ones have a wide to very wide distribution (cosmopolitan, southern hemisphere, ...), so that endemism of freshwater nematodes is very low, if it exists at all. This is due to easy dispersal capacities of nematodes on the one hand and the recent colonization (not more than 10 000, max. 40 000 years) on the other hand.     

Subfreezing Growth of the Sea Ice Bacterium “<Emphasis Type="Italic">Psychromonas ingrahamii</Emphasis>”

Psychromonas ingrahamii, named for John L. Ingraham, was isolated from sea ice from off Point Barrow, Alaska. This large rod-shaped bacterium belongs to the gamma-Proteobacteria. P. ingrahamii is a psychrophilic, heterotrophic bacterium that is gas vacuolate and nonmotile. P. ingrahamii is notable in that it grows at a temperature of –12°C with a generation time of 240 h. This is the lowest growth temperatures of any organism authenticated by a growth curve. Jim Staley has enjoyed exploring the diversity of the microbial world, especially that of the budding and prosthecate bacteria as well as polar microorganisms, alongside Peter Hirsch during many years of friendship and collaboration. Happy 75th, Peter!     

Freshwater or marine origin of the vertebrates?

1. Paleontological data indicate that the earliest recognizable vertebrate remains, bone fragments of Upper Cambrian and Lower Ordovician heterostracan fishes, were deposited in a marine situation. 2. Since these earliest fossils are sporadic in occurrence, from atypical marine deposits and since they only represent the full grown adult stage, the possibility of a freshwater developmental stage or estuarine habitat cannot be excluded. 3. The hagfishes, supposedly the most primitive of living vertebrates, are exclusively marine and possess an osmoregulatory strategy (monovalent ion levels nearly identical with sea-water with little capability of regulation) that is consonant with a strictly marine evolutionary history. Possibly, but less parsimoniously, this strategy and habitat could be secondarily derived. 4. The hagfish has a glomerular kidney, renal sodium reabsorption and branchial pumps for the uptake of sodium and chloride which are indicative, but not unequivocally diagnostic, of a freshwater ancestry. 5. A scenario in which the earliest vertebrate was anadromous, breeding in fresh water and migrating to the sea, is consistent with the paleontological data and with the physiology and life history of living 'primitive' fishes. It also leads to more coherent explanations for the origin of bone and for the evolution of vertebrate special senses than do alternative marine scenarios.     

A Mechanistic Investigation of the Algae Growth “Droop” Model

In this work a mechanistic explanation of the classical algae growth model built by M. R. Droop in the late sixties is proposed. We first recall the history of the construction of the "predictive" variable yield Droop model as well as the meaning of the introduced cell quota. We then introduce some theoretical hypotheses on the biological phenomena involved in nutrient storage by the algae that lead us to a "conceptual" model. Though more complex than Droop's one, our model remains accessible to a complete mathematical study: its confrontation to the Droop model shows both have the same asymptotic behavior. However, while Droop's cell quota comes from experimental bio-chemical measurements not related to intra-cellular biological phenomena, its analogous in our model directly follows our theoretical hypotheses. This new model should then be looked at as a re-interpretation of Droop's work from a theoretical biologist's point of view.     

Physiological Characterization of an Anaerobic Ammonium-Oxidizing Bacterium Belonging to the “Candidatus Scalindua” Group

The phylogenetic affiliation and physiological characteristics (e.g., K_s and maximum specific growth rate [μ_max]) of an anaerobic ammonium oxidation (anammox) bacterium, “Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.     

Effects of Dichloroethene Isomers on the Induction and Activity of Butane Monooxygenase in the Alkane-Oxidizing Bacterium “Pseudomonas butanovora”

We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium “Pseudomonas butanovora.” Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of ~20 nmol mg protein⁻¹ of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein⁻¹. Oxidation of similar amounts of each DCE isomer (~20 nmol mg protein⁻¹) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown “P. butanovora” did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.     

Critical Evaluation of the Volumetric “Bottle Effect” on Microbial Batch Growth

We have analyzed the impact of surface-to-volume ratio on final bacterial concentrations after batch growth. We examined six bottle sizes (20 to 1,000 ml) using three independent enumeration methods to quantify growth. We found no evidence of a so-called volumetric bottle effect, thus contradicting numerous previous reports.Microbial batch growth during confined incubation in bottles of various sizes is used daily in a broad variety of microbiological studies and methods, including bioassays such as the assimilable organic carbon (AOC) assay (6, 10, 18) and the analysis of pure culture or microbial community growth in freshwater (3, 11, 19, 20). In this context, “bottle effect” or “volume effect” is a term that has cropped up frequently in aquatic microbiology papers (e.g., references 12, 13, and 21) during the last 100 years to explain inexplicable phenomena and variations in results obtained from such batch growth studies. The uncertainty surrounding this apparent effect was clearly summarized in a recent paper by Pernthaler and Amann (16): “Such investigations are often plagued by the mysterious ‘bottle effect’, a hard-to-define concept that reflects the worry of whether phenomena observed in confined assemblages are nonspecific consequences of the confinement rather than a result of the planned manipulation.” The “bottle effect” alludes to an apparent reaction of bacteria to batchwise incubation in a confined environment, and this concept has intermittently been linked to influences on final cell concentrations (3) and grazing/bacterivory (13), a change in viability/activity parameters (9), a change in cultivability (5), and a change in population composition (1).The fact that microbiological processes during confined incubation differ from those in the environment is indisputable. However, a particular section of “bottle effect” literature focuses specifically on a volumetric “bottle effect”, where the above-mentioned effects are linked specifically to the size (or surface-to-volume ratio) of the incubation vessel (3, 8, 11-13, 15, 21). One of the oldest and best-known studies summarized clearly: “It will be observed that the densest bacterial populations appear in the bottles of water which offer the largest area of glass surface per unit volume of water” (21). This idea has established itself as dogma during the last century, with only a few differing opinions (4). However, precious little empirical data that actually quantify and explain the volumetric “bottle effect” are ever presented. In one example, Bischofberger et al. (3) observed that incubation of groundwater led to significantly more growth (about 2 log units) in small bottles (100 ml) than in big ones (10 liters). More often, however, the “bottle effect” is merely mentioned, as if it is self-explanatory and indisputable (2, 11, 12). In the present study, we took a simple but detailed look at the effect of bottle size on the outcome of short-term (<5-day) batch growth assays and compared the data critically to information in the literature and current opinion on this topic.Three batch growth experiments were conducted to assess the volumetric bottle effect on final cell concentrations after growth into stationary phase. Six different bottle sizes were used, covering the ranges most often reported in “bottle effect” literature. All glassware and Teflon-coated caps were cleaned comprehensively as described elsewhere (6) to remove any traces of organic carbon that might have been present on surfaces. The bottle sizes were as follows (water volumes and surface area-to-volume ratios [square centimeters to milliliters] are respectively included in parentheses): 1,000 ml (900 ml, 0.3:1), 500 ml (400 ml, 0.4:1), 250 ml (200 ml, 0.6:1), 100 ml (90 ml, 0.8:1), 40 ml (35 ml, 1.5:1), and 20 ml (15 ml, 2.4:1). In the first experiment, a sample of natural river water (dissolved organic carbon [DOC], 3.8 mg/liter; AOC, 0.3 mg/liter) from a small oligotrophic stream was obtained, filter sterilized with a 50-kDa dialysis filter (Fresenius Medical Care), and inoculated (at 10³ cells/ml) with a microbial community used for AOC assays (19). In the second experiment, a sample of the effluent (DOC, 1.2 mg/liter; AOC, 0.03 mg/liter; total cell concentration [TCC], 3 × 10⁵ cells/ml) from a granulated active carbon filter situated in a drinking water pilot plant (7) was collected and used directly for the experiment without additional treatment or inoculation. For the third experiment, sterile Luria-Bertani (LB) medium (diluted 1:10,000; DOC, 0.7 mg/liter; AOC, 0.46 mg/liter) was inoculated with Vibrio cholerae O1 (10³ cells/ml) as described previously (19). The water from each experiment was distributed into triplicate flasks of each size and incubated (at 30°C) until stationary phase was reached. Stationary phase was indicated by no significant increase in the TCC (measured after 3, 4, and 5 days) on consecutive days. Samples from all experiments were analyzed (i) for TCCs after being stained with SYBR green I and subjected to flow cytometry (7, 19), (ii) for ATP by using a commercial luciferin-luciferase assay (Promega Corporation) (7), and (iii) for heterotrophic plate counts (HPC) on R2A agar by a pour plate method with incubation at 30°C for 10 days. Possible biofilm growth was checked by applying sonication to selected samples. However, no wall growth in bottles of any size was observed.Growth was observed in all three experiments. The results show the net growth after subtraction of the initial cell/ATP/HPC concentrations from the final concentrations (Fig. ​(Fig.1).1). The proposed concept of the volumetric bottle effect implies that more growth should occur in smaller bottles. All data sets were subjected to regression analysis, and we observed no significant correlation (P < 0.01) between bottle size and final growth in any of the experiments by any of the three independent methods used for quantification. Figure ​Figure1A1A shows the batch growth results for a natural microbial community in prefiltered river water. This experimental setup is reflective of a typical AOC assay (6) or batch cultivation of natural microbial communities (20). Figure ​Figure1B1B shows the results for direct incubation of a treated drinking water sample. This sample and experimental setup were chosen specifically to assess any potential volumetric “bottle effect” on an indigenous microbial community in a biologically stable water sample, where only limited growth is expected. Indeed, the final cell concentration in the sample was only about 25% higher than the original cell concentration. The cultivability (HPC/TCC × 100) at day 0 was 0.4%, and at the end of the experimental period it had increased to 2.5%. This points to increased cultivability as a result of growth during confinement (5), yet it does not relate at all to the size of the incubation vessel. Figure ​Figure1C1C shows the data for V. cholerae grown in sterile LB medium (diluted 1:10,000) to stationary phase. Again, this particular setup is of specific relevance since a recently published paper on the growth of V. cholerae referred directly to the volumetric “bottle effect” to explain rather large differences between growth results from two separate studies (11, 19). The data from Fig. ​Fig.1C1C suggest at least that a “bottle effect” should be ruled out as an interfering factor in this case.Open in a separate windowFIG. 1.Effects of bottle size on bacterial batch growth of a natural microbial community in filter-sterilized surface water (A), growth of bacteria during direct incubation of water from a drinking water treatment plant (B), and batch growth of a V. cholerae pure culture in diluted LB medium (C). Growth (expressed as the net growth) was quantified by flow cytometric total cell counting (circles), total ATP analysis (diamonds), and conventional plating (squares). All data points represent averages of triplicate measurements.The results presented in this study clearly dispute the concept of a volumetric “bottle effect” on the outcome of short-term batch growth assays, be it for pure cultures or natural microbial communities. These findings contradict evidence reported by many other researchers (3, 8, 11-13, 15, 21). Although the volumetric “bottle effect” is often cited as a somewhat mysterious occurrence, it is imperative that clear experimental data are required for the critical appraisal thereof. The main experimental theory behind the phenomenon is that organic carbon adsorbs to clean glass surfaces, thus locally concentrating the carbon and creating more favorable growth conditions (2, 14). This adsorption and the fact that bacteria can utilize such adsorbed carbon have been demonstrated experimentally (14). What has, in our opinion, not been shown conclusively is that these effects can be so dramatic that they would alter the growth of samples to the extent that different sizes of bottles would render different final cell numbers after growth. Since we have not observed any volumetric “bottle effect” in our work, we can only speculate on the possible reasons why this has been observed previously. One explanation may be that glassware contaminated with organic carbon can contribute to the perception of a volumetric “bottle effect,” as large surface-to-volume ratios (found in small bottles) would account for increased contamination compared to that in bottles with smaller ratios. Hence, more additional available carbon would be introduced into smaller bottles, giving rise to higher final cell numbers after growth. In this context, it is essential that a comprehensive glassware-cleaning protocol be followed, including heating to a high temperature (>500°C) and storage away from volatile organics (6). In addition, it is important that such experiments at low carbon concentrations are complemented with the inclusion of correct and sensitive controls to assess potential organic carbon contamination. For example, the use of deionized water as a negative control should be avoided, since the absence of inorganic nutrients is bound to lead to no growth and thus false-negative results (10). A good negative control would be water that is only carbon limited, e.g., bottled drinking water (17). Moreover, the use of multiple tools for analyzing growth, including cultivation-independent methods, is encouraged.In conclusion, we did not observe evidence of a volumetric bottle effect on short-term (<5-day) batch incubations. The findings of this study suggest that reference to the so-called volumetric bottle effect should be considered carefully unless supported by clear experimental data. This study does not dispute the fact that many authors have observed results implying apparent bottle effects during growth studies, but it questions the interpretation and understanding of this concept and the random use of the term “bottle effect” to explain uncertainty in results, specifically in relation to bottle size. Hopefully, these data will assist with experimental setups and comparison of data among different groups and stimulate discussion of and future research on this interesting, but slightly controversial, topic.     

Does the Antibacterial Activity of Silver Nanoparticles Depend on the Shape of the Nanoparticle? A Study of the Gram-Negative Bacterium Escherichia coli

In this work we investigated the antibacterial properties of differently shaped silver nanoparticles against the gram-negative bacterium Escherichia coli, both in liquid systems and on agar plates. Energy-filtering transmission electron microscopy images revealed considerable changes in the cell membranes upon treatment, resulting in cell death. Truncated triangular silver nanoplates with a {111} lattice plane as the basal plane displayed the strongest biocidal action, compared with spherical and rod-shaped nanoparticles and with Ag⁺ (in the form of AgNO₃). It is proposed that nanoscale size and the presence of a {111} plane combine to promote this biocidal property. To our knowledge, this is the first comparative study on the bactericidal properties of silver nanoparticles of different shapes, and our results demonstrate that silver nanoparticles undergo a shape-dependent interaction with the gram-negative organism E. coli.     

Plant Growth–Promoting Bacteria Facilitate the Growth of the Common Reed Phragmites australisin the Presence of Copper or Polycyclic Aromatic Hydrocarbons

To test whether plant growth–promoting bacteria might be useful in facilitating the growth of Phragmites australis, the common reed, in the presence of metals and organic compounds, P. australis seeds were treated with plant growth–promoting bacteria. The bacterium Pseudomonas asplenii AC was genetically transformed to express a bacterial gene encoding the enzyme 1-aminocyclopropane-1-carboxylate deaminase, and both the native and transformed bacteria were tested in conjunction with P. australis. Inoculation of seeds, which were subsequently grown in the presence of copper or creosote, with transformed P. asplenii AC significantly increased seed germination. Moreover, the addition of either native or transformed P. asplenii AC to P. australis seeds enabled the plants (shoots and roots) to attain a greater size than noninoculated plants after growth in soil in the presence of either copper or creosote.     

Laboratory Simulation of “Proteolytic Bacterium–Cyanobacterium” Interaction in Alkaliphilic Microbial Community

Paleontological Journal - The work continues the series of long-term studies of the microbial diversity of soda lakes and concerns the problem of anaerobic decomposition of proteins. The physiology...