Interactions of chromium with microorganisms and plants.

Chromium is a highly toxic non-essential metal for microorganisms and plants. Due to its widespread industrial use, chromium (Cr) has become a serious pollutant in diverse environmental settings. The hexavalent form of the metal, Cr(VI), is considered a more toxic species than the relatively innocuous and less mobile Cr(III) form. The presence of Cr in the environment has selected microbial and plant variants able to tolerate high levels of Cr compounds. The diverse Cr-resistance mechanisms displayed by microorganisms, and probably by plants, include biosorption, diminished accumulation, precipitation, reduction of Cr(VI) to Cr(III), and chromate efflux. Some of these systems have been proposed as potential biotechnological tools for the bioremediation of Cr pollution. In this review we summarize the interactions of bacteria, algae, fungi and plants with Cr and its compounds.

The bifunctional enzyme chitosanase-cellulase produced by the gram-negative microorganism Myxobacter sp. AL-1 is highly similar to Bacillus subtilis endoglucanases.

The gram-negative bacterium Myxobacter sp. AL-1 produces chitosanase-cellulase activity that is maximally excreted during the stationary phase of growth. Carboxymethylcellulase zymogram analysis revealed that the enzymatic activity was correlated with two bands of 32 and 35 kDa. Ion-exchange-chromatography-enriched preparations of the 32-kDa enzyme were capable of degrading the cellulose fluorescent derivatives 4-methylumbelliferyl-beta-D-cellobioside and 4-methylumbelliferyl-beta-D-cellotrioside. These enzymatic preparations also showed a greater capacity at 70 degrees C than at 42 degrees C to degrade chitosan oligomers of a minimum size of six units. Conversely, the beta-1,4 glucanolytic activity was more efficient at attacking carboxymethylcellulose and methylumbelliferyl-cellotrioside at 42 degrees C than at 70 degrees C. The 32-kDa enzyme was purified more than 800-fold to apparent homogeneity by a combination of ion-exchange and molecular-exclusion chromatography. Amino-terminal sequencing indicated that mature chitosanase-cellulase shares more than 70% identity with endocellulases produced by strains DLG, PAP115, and 168 of the gram-positive microorganism Bacillus subtilis.

Spore photoproduct lyase operon (splAB) regulation during Bacillus subtilis sporulation: modulation of splB-lacZ fusion expression by P1 promoter mutations and by an in-frame deletion of splA.

EsigmaG-dependent transcription of the splAB operon in the forespore at stage III of Bacillus subtilis sporulation initiates from two promoters, P1 preceding splA (major) and P3 preceding splB (minor). To explore the possible role of splA in controlling splB-encoded spore photoproduct lyase expression, we measured beta-galactosidase from splB-lacZ fusions integrated at the SPbeta prophage locus which contained point mutations or deletions which either inactivated or physically removed P1 and/or splA. Paradoxically, inactivation of P1 by point mutation or its removal by deletion from upstream resulted in elevated beta-galactosidase expression of the resulting splB-lacZ fusion, as did an in-frame deletion of splA which left P1 and P3 intact;however, expression of all fusions remained sporulation specific and EsigmaG dependent.

Copper Resistance and Accumulation in the Zygomycete Mucor rouxii

Two copper-resistant (Copr) mutants, strains P1 and P3, were obtained from the dimorphic fungus Mucor rouxii. They were characterized as to their ability to take up copper in a growth medium supplemented with this metal ion. Detection of copper by linear sweep striping voltammetry in cell walls and in the cell wall-free fraction of disrupted cells revealed a higher content of the metal in both mutant fractions, as compared with those of the copper-sensitive (Cops) parental strain. Copper binding by M. rouxii growing cells was also studied through the use of a cytochemical method based on the compounds neocuproine (NCP) and sodium diethyldithiocarbamate (DTC). This method indicated that the P1 Copr strain accumulated more metal than the parental Cops strain, both on the cellular surface and in the intracellular milieu.

Genetic evidence for independence between fermentative metabolism (ethanol accumulation) and yeast-cell development in the dimorphic fungus Mucor rouxii.

Three allyl-alcohol-resistant mutants were isolated in the dimorphic fungus Mucor rouxii and characterized with regard to their alcohol dehydrogenase (ADH) activity in vitro and in vivo as well as their ability to execute the morphological alternatives of dimorphism under different environmental stimuli, either in the absence or in the presence of oxygen. These studies indicated that fermentation and yeast-cell development are independent events and that ADH activity is essential for growth of the fungus in the absence of oxygen. Heterokaryon construction and analysis indicated that in the three mutant strains the corresponding genetic alterations are recessive nuclear mutations which behave as allelic in complementation tests.

Temporal regulation and forespore-specific expression of the spore photoproduct lyase gene by sigma-G RNA polymerase during Bacillus subtilis sporulation.

Bacterial spores are highly resistant to killing by UV radiation and exhibit unique DNA photochemistry. UV irradiation of spore DNA results in formation of spore photoproduct (SP), the thymine dimer 5-thyminyl-5,6-dihydrothymine. Repair of SP occurs during germination of Bacillus subtilis spores by two distinct routes, either by the general nucleotide excision repair (uvr) pathway or by a novel SP-specific monomerization reaction mediated by the enzyme SP lyase, which is encoded by the spl gene. Repair of SP occurs early in spore germination and is independent of de novo protein synthesis, suggesting that the SP repair enzymes are synthesized during sporulation and are packaged in the dormant spore. To test this hypothesis, the expression of a translational spl-lacZ fusion integrated at the spl locus was monitored during B. subtilis growth and sporulation. beta-Galactosidase expression from the spl-lacZ fusion was silent during vegetative growth and was not DNA damage inducible, but it was activated at morphological stage III of sporulation specifically in the forespore compartment, coincident with activation of expression of the stage III marker enzyme glucose dehydrogenase. Expression of the spl-lacZ fusion was shown to be dependent upon the sporulation-specific RNA polymerase containing the sigma-G factor (E sigma G), as spl-lacZ expression was abolished in a mutant harboring a deletion in the sigG gene and restored by expression of the sigG gene in trans. Primer extension analysis of spl mRNA revealed a major extension product initiating upstream from a small open reading frame of unknown function which precedes spl, and it revealed two other shorter minor extension products. All three extension products were present in higher quantities during sporulation and after sigG induction. The three putative transcripts are all preceded by sequences which share homology with the consensus sigma-G factor-type promoter sequence, but in vitro transcription by purified sigma-G RNA polymerase was detected only from the promoter corresponding to the major extension product. The open reading frame-spl operon therefore appears to be an additional member of the sigma-G regulon, which also includes as members the small, acid-soluble spore proteins which are in large part responsible for spore DNA photochemistry. Therefore, sporulating bacteria appear to coordinately regulate genes whose products not only alter spore DNA photochemistry but also repair the major spore-specific photoproduct during germination

Copper resistance mechanisms in bacteria and fungi.

Copper is both an essential micronutrient and a toxic heavy metal for most living cells. The presence of high concentrations of cupric ions in the environment promotes the selection of microorganisms possessing genetic determinants for copper resistance. Several examples of chromosomal and plasmid copper-resistance systems in bacteria have been reported, and the mechanisms of resistance have started to be understood at the molecular level. Bacterial mechanisms of copper resistance are related to reduced copper transport, enhanced efflux of cupric ions, or copper complexation by cell components. Copper tolerance in fungi has also been ascribed to diverse mechanisms involving trapping of the metal by cell-wall components, altered uptake of copper, extracellular chelation or precipitation by secreted metabolites, and intracellular complexing by metallothioneins and phytochelatins; only the metallothionein chelation mechanism has been approached with molecular detail.

Nikkomycin-resistant mutants of Mucor rouxii: physiological and biochemical properties.

We isolated three nikkomycin-resistant mutants of the dimorphic fungus M. rouxii which were physiologically characterized regarding their response to yeast-phase inducing conditions and their sensitivity to bacilysin. Mutant strains G21 and G23, showed a qualitatively normal, though delayed, dimorphic transition and partial cross-resistance to bacilysin. Mutant strain G27 showed an altered dimorphism, producing a high proportion (50%) of hyphal cells, and a wild-type sensitivity to bacilysin. Cell-free extracts from this mutant exhibited an activity of both basal and protease-activated chitin synthetase which was overexpressed as compared with the parental strain and mutants G21 and G23. Results are discussed in terms of the different genetic background of the mutants.

Chitin synthetase activity in a developmental mutant of Phycomyces blakesleeanus.

Chitin synthetase activity was analyzed in vitro and in vivo in two morphogenetic stages, namely, dormant spore cells and germlings of the wild type strain and the developmental mutant S356 of Phycomyces blakesleeanus. In vitro experiments showed a much higher specific activity in dormant spores of the mutant strain than in those of the wild-type. This difference was restricted to the dormant spore phase since germlings exhibited comparable levels of activity to those detected in the wild-type strain. Although no correlation was observed between chitin synthesis in vitro and in vivo in mutant spores, germination of these cells was accompanied by an earlier expression of chitin synthetase in vivo. Germination of mutant spores in liquid medium produced morphologically aberrant germlings. Contrary to the extended mycelial growth of the wild-type strain in solid medium, the mutant grew with a typical colonial morphology. Results are discussed in relation to the possible basis of the mutant phenotype.

Altered expression of chitin synthetase activity and biochemical changes in the cell wall in a developmental mutant of Phycomyces.

The Phycomyces developmental mutant S356 elaborates spores which show a much poorer viability and a higher affinity for Calcofluor White than the wild-type spores. Protease-activated extracts of the mutant spores showed higher levels of chitin synthetase activity than the parental strain-derived spores. High levels of enzyme activity in the mutant extracts, but not in the corresponding wild-type extracts, could be detected in the absence of an exogenous protease. The high basal active chitin synthetase is not the result of activation by endogeneous proteases during cell breakage since protease inhibitors did not reduce, but rather increased, the activity levels. The analysis of cell wall composition in the mutant spores revealed significant changes in the proportion of uronic acids and protein but not in chitin. The mutant phenotype is discussed in relation to the developmental stage at which the alterations connected with cell wall metabolism occurred.