Gene transcript analysis of assimilatory iron limitation in Geobacteraceae during groundwater bioremediation.

Limitations on the availability of Fe(III) as an electron acceptor are thought to play an important role in restricting the growth and activity of Geobacter species during bioremediation of contaminated subsurface environments, but the possibility that these organisms might also be limited in the subsurface by the availability of iron for assimilatory purposes was not previously considered because copious quantities of Fe(II) are produced as the result of Fe(III) reduction. Analysis of multiple Geobacteraceae genomes revealed the presence of a three-gene cluster consisting of homologues of two iron-dependent regulators, fur and dtxR (ideR), separated by a homologue of feoB, which encodes an Fe(II) uptake protein. This cluster appears to be conserved among members of the Geobacteraceae and was detected in several environments. Expression of the fur-feoB-ideR cluster decreased as Fe(II) concentrations increased in chemostat cultures. The number of Geobacteraceae feoB transcripts in groundwater samples from a site undergoing in situ uranium bioremediation was relatively high until the concentration of dissolved Fe(II) increased near the end of the field experiment. These results suggest that, because much of the Fe(II) is sequestered in solid phases, Geobacter species, which have a high requirement for iron for iron-sulfur proteins, may be limited by the amount of iron available for assimilatory purposes. These results demonstrate the ability of transcript analysis to reveal previously unsuspected aspects of the in situ physiology of microorganisms in subsurface environments.

Subsurface clade of Geobacteraceae that predominates in a diversity of Fe(III)-reducing subsurface environments.

There are distinct differences in the physiology of Geobacter species available in pure culture. Therefore, to understand the ecology of Geobacter species in subsurface environments, it is important to know which species predominate. Clone libraries were assembled with 16S rRNA genes and transcripts amplified from three subsurface environments in which Geobacter species are known to be important members of the microbial community: (1) a uranium-contaminated aquifer located in Rifle, CO, USA undergoing in situ bioremediation; (2) an acetate-impacted aquifer that serves as an analog for the long-term acetate amendments proposed for in situ uranium bioremediation and (3) a petroleum-contaminated aquifer in which Geobacter species play a role in the oxidation of aromatic hydrocarbons coupled with the reduction of Fe(III). The majority of Geobacteraceae 16S rRNA sequences found in these environments clustered in a phylogenetically coherent subsurface clade, which also contains a number of Geobacter species isolated from subsurface environments. Concatamers constructed with 43 Geobacter genes amplified from these sites also clustered within this subsurface clade. 16S rRNA transcript and gene sequences in the sediments and groundwater at the Rifle site were highly similar, suggesting that sampling groundwater via monitoring wells can recover the most active Geobacter species. These results suggest that further study of Geobacter species in the subsurface clade is necessary to accurately model the behavior of Geobacter species during subsurface bioremediation of metal and organic contaminants.

Potential for quantifying expression of the Geobacteraceae citrate synthase gene to assess the activity of Geobacteraceae in the subsurface and on current-harvesting electrodes.

The Geobacteraceae citrate synthase is phylogenetically distinct from those of other prokaryotes and is a key enzyme in the central metabolism of Geobacteraceae. Therefore, the potential for using levels of citrate synthase mRNA to estimate rates of Geobacter metabolism was evaluated in pure culture studies and in four different Geobacteraceae-dominated environments. Quantitative reverse transcription-PCR studies with mRNA extracted from cultures of Geobacter sulfurreducens grown in chemostats with Fe(III) as the electron acceptor or in batch with electrodes as the electron acceptor indicated that transcript levels of the citrate synthase gene, gltA, increased with increased rates of growth/Fe(III) reduction or current production, whereas the expression of the constitutively expressed housekeeping genes recA, rpoD, and proC remained relatively constant. Analysis of mRNA extracted from groundwater collected from a U(VI)-contaminated site undergoing in situ uranium bioremediation revealed a remarkable correspondence between acetate levels in the groundwater and levels of transcripts of gltA. The expression of gltA was also significantly greater in RNA extracted from groundwater beneath a highway runoff recharge pool that was exposed to calcium magnesium acetate in June, when acetate concentrations were high, than in October, when the levels had significantly decreased. It was also possible to detect gltA transcripts on current-harvesting anodes deployed in freshwater sediments. These results suggest that it is possible to monitor the in situ metabolic rate of Geobacteraceae by tracking the expression of the citrate synthase gene.

The effect of processing and cryopreservation on nucleated umbilical cord blood cells.

Recovery of nucleated cord blood cells after storage in liquid nitrogen was evaluated. Red cells were depleted using Ficoll-Paque or Puregene red cell lyses. Freeze Medium contained 10% dimethylsulfoxide and 20% serum for cryoprotection. Recovery of the original cell population remaining serviceable for fluorescence activated cell sorting (FACS) was 12 +/- 10% (average +/- standard deviation), with a range of 1% to 55%. Viability measured by FACS analysis after freezing was significantly lower than that of the same specimens prior to freezing, 62 +/- 20% compared to 91+/-11% (p<0.001). Percentage CD45+34+ cells were the same for fresh and frozen cells. Gestational age at which specimens were collected had no effect on the percent cells carrying the CD45+34+ markers. We conclude that better cryoprotective supplements are needed to insure consistent high recovery of viable nucleated umbilical cord blood cells after preservation in liquid nitrogen.

Prevalence of Mycoplasma bacteria in amniotic fluid at the time of genetic amniocentesis using the polymerase chain reaction.

OBJECTIVE: To use the polymerase chain reaction (PCR) to detect the presence of bacteria and Mycoplasma in amniotic fluid at the time of genetic amniocentesis and to assess their relationship to amniotic fluid interleukin-6 (IL-6) concentration and preterm delivery. STUDY DESIGN: A prospective study was performed on 78 asymptomatic pregnancies presenting for genetic amniocentesis. Amniotic fluid samples were analyzed by PCR for the detection of bacterial 16S ribosomal RNA, by PCR/enzyme-linked immunosorbent assay (ELISA) for Mycoplasma and by ELISA analysis for IL-6 concentration. RESULTS: All 78 samples were analyzed, and bacterial RNA was detected in 18% of them. However, no sample tested positive for Mycoplasma. The mean IL-6 concentration was 435 pg/mL. There was no difference in IL-6 levels or gestational age between bacteria-positive and -negative groups. CONCLUSION: Amniotic fluid may not be sterile in the midtrimester, yet the presence of bacteria, as detected by PCR, does not always result in an inflammatory response or adverse perinatal outcome.