An empirical strategy for characterizing bacterial proteomes across species in the absence of genomic sequences

Global protein identification through current proteomics methods typically depends on the availability of sequenced genomes. In spite of increasingly high throughput sequencing technologies, this information is not available for every microorganism and rarely available for entire microbial communities. Nevertheless, the protein-level homology that exists between related bacteria makes it possible to extract biological information from the proteome of an organism or microbial community by using the genomic sequences of a near neighbor organism. Here, we demonstrate a trans-organism search strategy for determining the extent to which near-neighbor genome sequences can be applied to identify proteins in unsequenced environmental isolates. In proof of concept testing, we found that within a CLUSTAL W distance of 0.089, near-neighbor genomes successfully identified a high percentage of proteins within an organism. Application of this strategy to characterize environmental bacterial isolates lacking sequenced genomes, but having 16S rDNA sequence similarity to Shewanella resulted in the identification of 300-500 proteins in each strain. The majority of identified pathways mapped to core processes, as well as to processes unique to the Shewanellae, in particular to the presence of c-type cytochromes. Examples of core functional categories include energy metabolism, protein and nucleotide synthesis and cofactor biosynthesis, allowing classification of bacteria by observation of conserved processes. Additionally, within these core functionalities, we observed proteins involved in the alternative lactate utilization pathway, recently described in Shewanella.     

Engineering Deinococcus geothermalis for bioremediation of high-temperature radioactive waste environments

Deinococcus geothermalis is an extremely radiation-resistant thermophilic bacterium closely related to the mesophile Deinococcus radiodurans, which is being engineered for in situ bioremediation of radioactive wastes. We report that D. geothermalis is transformable with plasmids designed for D. radiodurans and have generated a Hg(II)-resistant D. geothermalis strain capable of reducing Hg(II) at elevated temperatures and in the presence of 50 Gy/h. Additionally, D. geothermalis is capable of reducing Fe(III)-nitrilotriacetic acid, U(VI), and Cr(VI). These characteristics support the prospective development of this thermophilic radiophile for bioremediation of radioactive mixed waste environments with temperatures as high as 55 degrees C.     

Shifts in archaeal communities associated with lithological and geochemical variations in subsurface Cretaceous rock

Subsurface microbial community structure in relation to geochemical gradients and lithology was investigated using a combination of molecular phylogenetic and geochemical analyses. Discreet groundwater and substratum samples were obtained from depths ranging from 182 to 190 m beneath the surface at approximately 10-cm intervals using a multilevel sampler (MLS) that straddled Cretaceous shale and sandstone formations at a site in the southern San Juan Basin in New Mexico. DNA and RNA were extracted directly from quartzite sand substratum loaded into individual cells of the MLS and colonized in situ. Polymerase chain reaction (PCR)-mediated T-RFLP analysis of archaeal rRNA genes (rDNA) in conjunction with partial sequencing analysis of archaeal rDNA libraries and quantitative RNA hybridization with oligonucleotide probes were used to probe community structure and function. Although total microbial populations remained relatively constant over the entire depth interval sampled, significant shifts in archaeal populations, predominantly methanogens, were observed. These shifts coincided with the geochemical transition from relatively high methane (26 mM), low sulphate (< 3 mg l(-1)) conditions in the region adjacent to the organic matter-rich shale to relatively low-methane (< 0.5 mM), high-sulphate (48 mg l(-1)) conditions in the organic-poor sandstone beneath the shale. These results indicated that active, phylogenetically diverse archaeal communities were present in the subsurface Cretaceous rock environment at this site and that major archaeal clades shifted dramatically over scales of tens of centimetres, corresponding to changes in the lithology and geochemical gradients.     

Laser-induced fluorescence (LIF) spectra of herbaceous and woody pre- and post-digested plant material

Filtrate from pre- and post-digested plant material was exposed to 355-nm pulsed laser light and the subsequent laser-induced fluorescence (LIF) was recorded. Similarities and differences among spectra from 20 materials are discussed. Each material was replicated once, dried, ground, and exposed to chloroform (CHCl₃) for 24 h. The material represented aged (1 to 18 years old) plants from different herbaceous (grasses and forbs) and woody plant life forms. Mean peak fluorescence recorded among materials differed (P<0.0001) in both wavelength and peak amplitude (counts) across the spectral range (387 to 788 nm). Peak fluorescence was evaluated within each of three arbitrary color categories, blue near 455 nm and red near 674 nm, while only 16 of the materials produced a green peak near 528 nm. In general, the blue and green fluorescence peaks were broad while the red peak was narrow. Mean peak counts were largest in the red range. Varying amounts of laser beam absorption occurred among the materials evaluated due to different concentrations of filtrate and different absorption efficiencies; therefore, amplitude data (counts) were not used to determine statistical differences among materials. To overcome difficulties attributed to the raw count data, red/blue, red/green and blue/green count ratios within replicates were calculated. Using all three count ratios in a multivariate analysis of variance, the 16 materials could be separated into nine different (P<0.05) material groupings. The LIF technique may provide a reliable means to separate ground pre- and post-digested plant materials following further research into determining what fluorophores are producing the spectral signatures and how sample preparation affect peak wavelengths.     

Feeding heterogeneity in ciliate populations: Effects of culture age and nutritional state

We have used a novel approach in conjunction with flow cytometry to quantify the biological heterogeneity of populations of the ciliate Tetrahymena pyriformis. It was found that the rate of particle uptake of exponentially growing cells is not uniform among cells and partially correlated with cell size. The physiological state and growth history of the culture was found to affect to a large degree the population's feeding heterogeneity. Stationary phase populations exhibited more uniform feeding behavior, as cell aging affects all cells and effectively reduces their ability to feed. Cells that were removed from the growth medium and resuspended in nonnutritive medium exhibited a more heterogeneous feeding behavior. The starved cells were stimulated to feed at considerably higher rates, and the stimulatory effect was more pronounced for larger cells. It is therefore demonstrated that population heterogeneity has to be evaluated in conjunction with the populations growth state as it is determined by the history of the population's growth and nutritional state. (c) 1994 John Wiley & Sons, Inc.     

Effects of attachment of bacteria to chemostat walls in a microbial predator-prey relationship

Mixed cultures of the protozoan Tetrahymena pyriformis and the bacterium Escherichia coli were propagated in chemostats fed with glucose and minimal mineral salts medium. The interior surfaces of some chemostats were treated with a silicone compound but those of other chemostats were not. Data obtained showed that bacteria but not protozoans were attached strongly to the walls of the chemostats, that silicone treatment reduced the density of the attached bacteria by two orders of magnitude or more, and that the presence of the attached bacteria had significant effects on the dynamics of the microbial predator-prey system. Attempts were made to simulate the data by various mathematical models. It was found that a model based on combination of the Topiwala-Hamer model for wall attachment and a multiple saturation model for growth of the protozoans on the bacteria gave a reasonable fit of all the data.     

Reduction of Fe(III), Cr(VI), U(VI), and Tc(VII) by Deinococcus radiodurans R1

Deinococcus radiodurans is an exceptionally radiation-resistant microorganism capable of surviving acute exposures to ionizing radiation doses of 15,000 Gy and previously described as having a strictly aerobic respiratory metabolism. Under strict anaerobic conditions, D. radiodurans R1 reduced Fe(III)-nitrilotriacetic acid coupled to the oxidation of lactate to CO(2) and acetate but was unable to link this process to growth. D. radiodurans reduced the humic acid analog anthraquinone-2,6-disulfonate (AQDS) to its dihydroquinone form, AH(2)DS, which subsequently transferred electrons to the Fe(III) oxides hydrous ferric oxide and goethite via a previously described electron shuttle mechanism. D. radiodurans reduced the solid-phase Fe(III) oxides in the presence of either 0.1 mM AQDS or leonardite humic acids (2 mg ml(-1)) but not in their absence. D. radiodurans also reduced U(VI) and Tc(VII) in the presence of AQDS. In contrast, Cr(VI) was directly reduced in anaerobic cultures with lactate although the rate of reduction was higher in the presence of AQDS. The results are the first evidence that D. radiodurans can reduce Fe(III) coupled to the oxidation of lactate or other organic compounds. Also, D. radiodurans, in combination with humic acids or synthetic electron shuttle agents, can reduce U and Tc and thus has potential applications for remediation of metal- and radionuclide-contaminated sites where ionizing radiation or other DNA-damaging agents may restrict the activity of more sensitive organisms.     

Development of a vital fluorescent staining method for monitoring bacterial transport in subsurface environments

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10(5) CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.