Low-substrate regulated microaerophilic behavior as a stress response of aquatic and soil bacteria.

Low-substrate regulated microaerophilic behavior (LSRMB) was observed in 10-54% of the bacteria isolated from several fresh-water lakes or ponds, subsurface soils, activated sludge, and Antarctic dry valley soils. Five Pseudomonas and two Bacillus type species showed LSRMB. A subsurface Pseudomonas jessenii strain was used as a model to show the metabolic interaction between substrate and oxygen concentrations, cell band movement, and the appearance of unique stress lipids and proteins. When the oxygen in the P. jessenii culture medium was increased from 11% to 100% saturation under atmospheric condition, the concentration of 17:0 cyclopropane fatty acid, a stress indicator, increased five-fold, and four unique proteins were also detected. This stress response occurred only in low-substrate media. It is our hypothesis that LSRMB is a common but under-appreciated trait of many aquatic and soil bacteria.

Determining chemotactic responses by two subsurface microaerophiles using a simplified capillary assay method.

A simplified capillary chemotaxis assay utilizing a hypodermic needle, syringe, and disposable pipette tip was developed to measure bacterial tactic responses. The method was applied to two strains of subsurface microaerophilic bacteria. This method was more convenient than the Adler method and required less practice. Isolate VT10 was a strain of Pseudomonas syringae, which was isolated from the shallow subsurface. It was chemotactically attracted toward dextrose, glycerol, and phenol, which could be used as sole carbon sources, and toward maltose, which could not be used. Isolate MR100 was phylogenetically related to Pseudomonas mendocina and was isolated from the deep subsurface. It showed no tactic response to these compounds, although, it could use dextrose, maltose, and glycerol as carbon sources. The chemotaxis results obtained by the new method were verified by using the swarm plate assay technique. The simplified technique may be useful for routine chemotactic testing.

Survival of subsurface microorganisms exposed to UV radiation and hydrogen peroxide.

Aerobic and microaerophilic subsurface bacteria were screened for resistance to UV light. Contrary to the hypothesis that subsurface bacteria should be sensitive to UV light, the organisms studied exhibited resistance levels as efficient as those of surface bacteria. A total of 31% of the aerobic subsurface isolates were UV resistant, compared with 26% of the surface soil bacteria that were tested. Several aerobic, gram-positive, pigmented, subsurface isolates exhibited greater resistance to UV light than all of the reference bacterial strains tested except Deinococcus radiodurans. None of the microaerophilic, gram-negative, nonpigmented, subsurface isolates were UV resistant; however, these isolates exhibited levels of sensitivity similar to those of the gram-negative reference bacteria Escherichia coli B and Pseudomonas fluorescens. Photoreactivation activity was detected in three subsurface isolates, and strain UV3 exhibited a more efficient mechanism than E. coli B. The peroxide resistance of four subsurface isolates was also examined. The aerobic subsurface bacteria resistant to UV light tolerated higher levels of H2O2 than the microaerophilic organisms. The conservation of DNA repair pathways in subsurface microorganisms may be important in maintaining DNA integrity and in protecting the organisms against chemical insults, such as oxygen radicals, during periods of slow growth.

Anaerobic degradation of cyanuric Acid, cysteine, and atrazine by a facultative anaerobic bacterium.

A facultative anaerobic bacterium that rapidly degrades cyanuric acid (CA) was isolated from the sediment of a stream that received industrial wastewater effluent. CA decomposition was measured throughout the growth cycle by using a high-performance liquid chromatography assay, and the concomitant production of ammonia was also measured. The bacterium used CA or cysteine as a major, if not the sole, carbon and energy source under anaerobic, but not aerobic, conditions in a defined medium. The cell yield was greatly enhanced by the simultaneous presence of cysteine and CA in the medium. Cysteine was preferentially used rather than CA early in the growth cycle, but all of the CA was used without an apparent lag after the cysteine was metabolized. Atrazine was also degraded by this bacterium under anaerobic conditions in a defined medium.

Rapid enumeration of Fecal Coliforms in water by a colorimetric beta-galactosidase assay.

The colorimetric beta-galactosidase assay is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-beta-D-galactoside (ONPG) by fecal coliforms. This technique provides an estimate of the fecal coliform concentration within 8 to 20 h. A 100-ml portion of test sample was passed through a 0.45 micrometer membrane filter. This filter was then incubated at 37 degrees C for 1 h in EC medium followed by the addition of filter-sterilized ONPG. The incubation was continued at 44.5 degrees C until a half-maximum absorbance (at 420 nm) was reached. The time between the start of incubation and the half-maximum absorbance was proportional to the concentration of fecal coliforms present. Escherichia coli (K-12) was used to measure the kinetics of substrate hydrolysis and the response time of different cell concentrations. High cell densities produced an immediate response, whereas 1 cell/ml will produce a response in less than 20 h. In field studies in which samples were taken from a range of grossly polluted streams to relatively clean lake water, a linear correlation between ONPG hydrolysis times and fecal coliform most-probable-number values was established. A total of 302 isolates randomly selected from positive ONPG-EC media, which were derived from 11 different habitats, were identified as E. coli (96.69 percent), Enterobacter cloacae (2.32 percent), Klebsiella pneumoniae (0.66 percent), and Citrobacter freundii (0.33 percent).

Manganese-mediated morphogenesis in Penicillium claviforme and Penicillium clavigerum.

The colonial morphology and growth of Penicillium claviforme and P. clavigerum in manganese-deficient, defined medium, were examined with and without manganese supplementation. In unsupplemented medium rendered free of manganese through treatment with a chelating resin, P. claviforme grew but did not produce coremia. Similar results were obtained for P. clavigerum on media devoid of added manganese. However, when these media were supplemented with optimal concentrations of manganese both fungi produced coremia. The presence of manganese in various colony sections oed in the conidia.

Heterofermentative carbohydrate metabolism of lactose-impaired mutants of Streptococcus lactis.

Two mutants of Streptococcus lactis ATCC 11454 have been isolated which possess an impaired lactose-fermenting capacity; galactose utilization is also affected, but to a lesser extent. Although the Embden-Meyerhof-Parnas pathway is the major, if not the sole, pathway of carbohydrate metabolism in the three strains, the fermentation end products of the mutants are dramatically different from the typical homolactic pattern of the wild type. Under conditions of low oxygen tension and growth-limiting lactose concentrations, mutant strain T-1 produces largely formic acid, acetic acid (2:1), and ethanol rather than lactic acid. Aerated cultures produce acetic acid, CO(2) (1:1), acetyl-methylcarbinol, and diacetyl. When the mutants use galactose as an energy source, lactic acid is the major end product, but significant heterofermentative activity is observed. The aberrations responsible for the mutant phenotypes reside in the proteins which catalyze the transport and hydrolysis of galactosides. It is hypothesized that the impaired transport system of the mutants reduces the intracellular pool of glycolytic intermediates below that of the wild type. Since fructose-1, 6-diphosphate is an activator of lactic dehydrogenase in S. lactis, lactic acid production is reduced, and pathways leading to the formation of other products are expressed.