[1] H 2 is probably the most important substrate for terrestrial subsurface lithoautotrophic microbial communities. Abiotic H 2 generation is an essential component of subsurface ecosystems truly independent of surface photosynthesis. Here we report that H 2 concentrations in fracture water collected from deep siliclastic and volcanic rock units in the Witwatersrand Basin, South Africa, ranged up to two molar, a value far greater than observed in shallow aquifers or marine sediments. The high H 2 concentrations are consistent with that predicted by radiolytic dissociation of H 2 O during radioactive decay of U, Th, and K in the host rock and the observed He concentrations. None of the other known H 2 -generating mechanisms can account for such high H 2 abundance either because of the positive free energy imposed by the high H 2 concentration or pH or because of the absence of required mineral phases. The radiolytic H 2 is consumed by methanogens and abiotic hydrocarbon synthesis. Our calculations indicate that radiolytic H 2 production is a ubiquitous and virtually limitless source of energy for deep crustal chemolithoautotrophic ecosystems.

Pulmonary infection by mucoid, alginate-producing Pseudomonas aeruginosa is the leading cause of mortality among patients suffering from cystic fibrosis. Alginate-producing P. aeruginosa is uniquely associated with the environment of the cystic fibrosis-affected lung, where alginate is believed to increase resistance to both the host immune system and antibiotic therapy. Recent evidence indicates that P. aeruginosa is most resistant to antibiotics when the infecting cells are present as a biofilm, as they appear to be in the lungs of cystic fibrosis patients. Inhibition of the protective alginate barrier with nontoxic compounds targeted against alginate biosynthetic and regulatory proteins may prove useful in eradicating P. aeruginosa from this environment. Our research has dealt with elucidating the biosynthetic pathway and regulatory mechanism(s) responsible for alginate synthesis by P. aeruginosa. This review summarizes reports on the role of alginate in cystic fibrosis-associated pulmonary infections caused by P. aeruginosa and provides details about the biosynthesis and regulation of this exopolysaccharide.

In Pseudomonas putida, the catBC operon encodes enzymes involved in benzoate degradation. Previous studies have determined that these enzymes are induced when P. putida is grown in the presence of benzoate. Pseudomonads are common soil bacteria which are capable of degrading many toxic compounds. These compounds are utilized as sources of nutrients after they are converted into intermediates common to conventional metabolic pathways. Understanding the regulation of genes which encode enzymes responsible for catabolism is of great interest to the field of biodegradation. Studies concerning the nature of substrate specificity for different catabolic enzymes may eventually lead to the engineering of novel catabolic pathways for compounds that currently cannot be degraded by nature. Since many of these recalcitrant compounds contain one or more aromatic rings (7,8,12), our laboratory has been studying the regulation of benzoate catabolism by Pseudomonas putida in order to better understand the regulation of the biodegradative processes.The chromosomal genes of P. putida which code for the enzymes that catabolize benzoate to 3-ketoadipate have been cloned and characterized (1, 2, 34, 35). Figure 1A shows that the catBC operon encodes the enzymes cis,cismuconate lactonizing enzyme and muconolactone isomerase, respectively. Early studies demonstrated that growth on benzoate increased the levels of all 3-ketoadipate enzymes (20,22,34,35 considerable homology to the LysR family of regulatory proteins (13, 24).In an earlier study (25), our laboratory reported that catR and catBC are divergently transcribed with overlapping promoters (Fig. 1B). It was also reported that CatR binds to a single 27-bp site located in the region between the catR and catBC genes (Fig. 1C). In this study, we have examined the mechanism by which CatR activates the catBC operon. Gel shift studies have determined that cis,cis-muconate increases the binding affinity of CatR for the catBC promoter region. In addition, cis,cis-muconate allows CatR to bind to a second site of the catBC promoter region, thereby activating catBC transcription. MATERIALS AND METHODSBacterial strains, plasmids, and media. The Escherichia coli strain used for general cloning procedures was JM109[A(pro-lac) recAl thi-1 supE endA gyrA96 hsdR relA1(F' traD36 proAB lacIq lacZAM15)] (21). JM109 was grown at 37°C in Luria broth (LB) (19). Agar was added to 1.5% for plates. Antibiotics were added to sterile media in the following concentrations: ampicillin, 75 ,g/ml; and kanamycin, 75 ,g/ml.Purification of CatR. The CatR protein was overexpressed in JM109 by using the expression vector pKR119AHf as described previously (25). Cells grown overnight in 500 ml of LB supplemented with ampicillin were added to 6 liters of the same medium and allowed to grow for 1 h at 37°C. At this time, isopropyl-13-D-thiogalactopyranoside (IPTG; final concentration, 1 mM) was added to the culture, after which growth was allowed to continue for an additional 10 h. The 7798 on June 7, 2019 by guest

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/SEstained 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.