The microbial degradation of azo dyes: minireview.

The removal of dyes in wastewater treatment plants still involves physical or chemical processes. Yet numerous studies currently exist on degradation based on the use of microbes-which is a well-studied field. However progress in the use of biological methods to deal with this environmentally noxious waste is currently lacking. This review focuses on the largest dye class, that is azo dyes and their biodegradation. We summarize the bacteria identified thus far which have been implicated in dye decolorization and discuss the enzymes involved and mechanisms by which these colorants are broken down.

Identification of a gene responsible for amido black decolorization isolated from Amycolatopsis orientalis.

Investigation into the biodegradative capability of several actinomycetes led to the discovery of many strains possessing an ability to decolorize a variety of azo and triphenylmethane dyes. Of particular interest is an Amycolatopsis sp. isolate which displayed the ability to decolorize the azo dye amido black. Through the screening of a DNA library a 4.5 kbp fragment coding for the potential decolorization gene was identified. The sequencing of this gene fragment led to the prediction of seven open reading frames encoding a polyprenyl synthetase, cupin-2 conserved barrel domain, transcriptional regulator, membrane protein, DNA-damage inducible gene and two hypothetical proteins. A gene coding for a protein consisting of 312 amino acids with 77 % similarity to a conserved hypothetical protein in Amycolatopsis mediterranei was tentatively identified as the enzyme of interest. This is the first report of an amido black biodegrading gene identified in this species.

Structures of ADP-ribosylated rifampicin and its metabolite: intermediates of rifampicin-ribosylation by Mycobacterium smegmatis DSM43756.

23-(O-ADP-Ribosyl)rifampicin [RIP-TAs (3, Na+ form), RIP-TAf (4, H+ form)] was obtained as an intermediate in the conversion process of rifampicin (1) to RIP-Mb (2) that is mediated by cell homogenates of Mycobacterium smegmatis DSM43756 or of Escherichia coli carrying a mycobacterial mono(ADP-ribosyl) transferase gene, in the presence of NADH. 23-[O-(5'-Phosphoribosyl)]rifampicin (5, RIP-TAp) was also obtained by the reaction of rifampicin with NADH in the presence of a homogenate of M. smegmatis. The structures of 3, 4, and 5 were determined by means of MS and NMR analyses.

Light inhibits rifampicin inactivation and reduces rifampicin resistance due to a cloned mycobacterial ADP-ribosylation gene.

Rifampicin is a principal drug used to combat infections by mycobacteria and related organisms. Most strains of Mycobacterium are able to inactivate this antibiotic by ribosylation via an ADP-ribosylated intermediate. We found that this inactivation was inhibited by light at levels similar to those prevailing in laboratory environments. Rifampicin resistance arising from the cloned ADP-ribosyl transferase was also greatly diminished at these light levels. The cloned Rhodococcus equi monooxygenase which inactivates this antibiotic by a different mechanism was, in contrast, not inhibited by light.

ADP-ribosylation as an intermediate step in inactivation of rifampin by a mycobacterial gene.

Mycobacterium smegmatis DSM43756 inactivates rifampin, and the inactivated antibiotic product recovered from culture medium was ribosylated on the 23-OH group. To study this process, the gene responsible for the inactivation was expressed at high levels by the lac promoter in Escherichia coli conferring resistance to >500 microg of antibiotic per ml. Cell homogenates generated a novel derivative designated RIP-TAs; in this study, we determined that RIP-TAs is 23-(O-ADP-ribosyl)rifampin. Our results indicated that RIP-TAs is an intermediate in the pathway leading to ribosylated rifampin and that the previously characterized gene encodes a mono(ADP-ribosyl)transferase which, however, shows no sequence similarity to other enzymes of this class.

Identification and characterization of a new intermediate in the ribosylative inactivation pathway of rifampin by Mycobacterium smegmatis.

Mycobacterium smegmatis DSM 43756 inactivates rifampin by ribosylation. To study this process of rifampicin, all possible inactivated forms of the antibiotic were extracted and purified. Structural studies showed the presence of a new inactivation product, designated RIP-TAp(23-phosphoribosyl-rifampin). Formation of 23-(O-ADP-ribosyl)rifampin (RIP-TAs) is the first step, followed by removal of AMP to give rise to the newly identified compound. Lastly, dephosphorylation leads to formation of 23-ribosyl-rifampin (RIP-Mb). Feeding experiments with the ADP-ribosylated antibiotic obtained from the cell homogenates of an Escherichia coli strain carrying the cloned M. smegmatis gene confirmed this rifampin inactivation process.

Cloning of genes that have environmental and clinical importance from rhodococci and related bacteria.

Generalised and specialised transduction systems were developed for Rhodococcus by means of bacteriophage Q4. The latter was used in conjunction with DNA from an unstable genetic element of R. rhodochrous to construct resistance plasmids which replicate in strains of R. equi, R. erythropolis and R. rhodochrous. One of the plasmids, pDA21, was joined with Erythropolis coli suicide vector pEcoR251 to obtain shuttle plasmids maintained in both rhodococci and E. coli. Conjugation between these rhodococcal strains demonstrated all were interfertile with each other and that some of the determinants for this were located on the unstable genetic element. Plasmids derived from this element, such as pDA21, carried the conjugative and self-incompatibility capacities; deletion analysis revealed that DNA necessary for self-incompatibility overlapped with that for arsenic resistance. Rifampicin is one of the principal chemotherapeutic agents used to treat infections by rhodococci and related organisms. The genes responsible for two types of inactivation have been cloned. The sequence of the R. equi DNA responsible for decomposition of the antibiotic strongly resembled those of monooxygenases acting upon phenolic compounds, consistent with the presence of a naphthalenyl moiety in the rifampicin molecule. Antibiotic resistance conferred by the gene was surprisingly specific to the semisynthetic compounds rifampicin (150-fold increase) and rifapentine (70-fold). Similar specificity was observed with the other inactivation gene cloned, which ribosylates rifampicin at the 23-hydroxyl position. A 60-bp sequence upstream of the monooxygenase and ribosylation genes is strikingly similar suggesting a shared pattern of regulation. Rhodococcal arsenic resistance and azo dye degradation genes have been cloned and characterised.

Ribosylative inactivation of rifampin by Mycobacterium smegmatis is a principal contributor to its low susceptibility to this antibiotic.

Mycobacterium smegmatis inactivates rifampin by ribosylating this antibiotic. The gene responsible for this ability was cloned and was shown to confer low-level resistance to this antibiotic (MIC increase, about 12-fold) in related organisms. A 600-bp subclone responsible for ribosylating activity and resistance carried an open reading frame of 429 bp. Targeted disruption of the gene in M. smegmatis resulted in mutants with much increased susceptibility to rifampin (MICs of 1.5 instead of 20 microg/ml) as well as the loss of antibiotic-inactivating ability. Also, disruption of this gene led to a much lower frequency of occurrence of spontaneous high-level rifampin-resistant mutants.

Monooxygenase-like sequence of a Rhodococcus equi gene conferring increased resistance to rifampin by inactivating this antibiotic.

A DNA clone from Rhodococcus equi conferring low-level rifampin resistance through the ability to inactivate this antibiotic via its decomposition was identified. The iri (inactivation of rifampin) gene consisted of an open reading frame of 1,437 bp encoding a 479-amino-acid sequence strongly resembling those of monooxygenases acting upon phenolic compounds or involved in polyketide antibiotic synthesis. When expressed in Escherichia coli, the gene conferred resistance to a > 50-micrograms/ml concentration of the drug.

Studies on the isopropylbenzene 2,3-dioxygenase and the 3-isopropylcatechol 2,3-dioxygenase genes encoded by the linear plasmid of Rhodococcus erythropolis BD2.

The enzymes responsible for the degradation of isopropylbenzene (IPB) and co-oxidation of trichloroethene (TCE) by Rhodococcus erythropolis BD2 are encoded by the linear plasmid pBD2. Fragments containing IPB catabolic genes were cloned from pBD2 and the nucleotide sequence was determined. By means of database searches and expression of the cloned genes in recombinant strains, we identified five clustered genes, ipbA1A2A3A4C, which encode the three components of the IPB 2,3-dioxygenase system, reductaseIPB (ipbA4), ferredoxinIPB (ipbA3) and the two subunits of the terminal dioxygenase (ipbA1A2), as well as the 3-isopropylcatechol (IPC) 2,3-dioxygenase (ipbC). The protein sequences deduced from the ipbA1A2A3A4C gene cluster exhibited significant homology with the corresponding proteins of analogous degradative pathways in Gram-negative and Gram-positive bacteria, but the gene order differed from most of them. IPB 2,3-dioxygenase and 3-IPC 2,3-dioxygenase could both be expressed in Escherichia coli, but the IPB 2,3-dioxygenase activities were too low to be detected by polarographic and TCE degradative means. However, inhibitor studies with the R. erythropolis BD2 wild-type are in accordance with the involvement of the IPB 2,3-dioxygenase in TCE oxidation.

Different rifampicin inactivation mechanisms in Nocardia and related taxa.

Mycolic acid-containing bacteria inactivate rifampicin in a variety of ways such as glucosylation, ribosylation, phosphorylation and decolorization. These inactivations were found to be a species-specific phenomena in Nocardia and related taxa. Gordona, Tsukamurella and fast-growing Mycobacterium modified rifampicin by ribosylation of the 23-OH group of the antibiotic. Such ribosylation was not observed in Rhodococcus and Corynebacterium, but phosphorylation of the 21-OH group of rifampicin was observed in one strain of Rhodococcus. Nocardia modified the antibiotic by glucosylation (23-OH group) and phosphorylation, but ribosylation was not observed.

Structure determination of ribosylated rifampicin and its derivative: new inactivated metabolites of rifampicin by mycobacterial strains.

Rifampicin (I) was converted into two inactivated products RIP-Ma and RIP-Mb by Mycobacterium smegmatis DSM43756. MS, NMR and chromatographic analysis showed the compounds to be 3-formyl-23-[O-(alpha-D-ribofuranosyl)]rifamycin SV (6) and 23-[O-(alpha-D-ribofuranosyl)]rifampicin (7), respectively.

Rifampicin inactivation by Bacillus species.

The ability of strains of Bacillus, Staphylococcus, Pseudomonas, and Escherichia coli to inactivate rifampicin was tested. Most Bacillus strains were found to inactivate rifampicin. Two modes of inactivation were identified; one was phosphorylation and the other involved decolorization. Presence or absence of either mechanism appeared unrelated to the phylogenetic relatedness of strains. None of the other organisms could inactivate this antibiotic.

Ribosylation by mycobacterial strains as a new mechanism of rifampin inactivation.

Several fast-growing Mycobacterium strains were found to inactivate rifampin. Two inactivated compounds (RIP-Ma and RIP-Mb) produced by these organisms were different from previously reported derivatives, i.e., phosphorylated or glucosylated derivatives, of the antibiotic. The structures of RIP-Ma and RIP-Mb were determined to be those of 3-formyl-23-[O-(alpha-D-ribofuranosyl)]rifamycin SV and 23-[O-(alpha-D-ribofuranosyl)]rifampin, respectively. To our knowledge, this is the first known example of ribosylation as a mechanism of antibiotic inactivation.

Identification of DNA involved in Rhodococcus chromosomal conjugation and self-incompatibility.

Genes essential for Rhodococcus chromosomal conjugation were found to be located on an unstable genetic element in one of the strains investigated. From this element two segments of DNA could be identified which were involved in conjugation of Rhodococcus strains. One region, spread over 8 kb, was involved in the property of self-incompatibility. A second region, of about 3 kb, was essential for conjugation between ATCC 12674 and related nocardioforms.

Nocardioform arsenic resistance plasmid characterization and improved Rhodococcus cloning vectors.

A representative of a group of related Rhodococcus arsenic resistance plasmids was characterized, locating the resistance genes and regions influencing host range and controlling copy number. This information, together with identification of antibiotic resistance determinants to replace the arsenic marker was used to construct Rhodococcus-Escherichia coli positive selection shuttle plasmids which, compared with those previously constructed, had half the size, a much higher copy number, and chloramphenicol rather than arsenic as selectable marker.

Cloning of DNA from a Rhodococcus strain conferring the ability to decolorize sulfonated azo dyes.

Azo dyes are recalcitrant pollutants. Two sulfonated azo dyes, Orange II and Amido black, are effectively decolorized by certain nocardioform strains of the genus Rhodococcus. A mutant of one of these strains was isolated which had lost azo-dye decolorizing ability and the strain was used to clone DNA conferring this ability, by screening a BclI library constructed from DNA of a decolorizing strain. The relevant genetic information was located on a 6.3-kb fragment of DNA.

Mutants lacking individual ribosomal proteins as a tool to investigate ribosomal properties.

We have isolated and characterized mutants which lack one or two of sixteen of the proteins of the Escherichia coli ribosome. The mutation responsible in each case mapped close to, and probably in, the corresponding gene. A conditional lethal phenotype and a variable degree of impairment in growth was observed. The missing protein was readily restored to the organelle if E coli or other eubacterial ribosomal proteins were added to a suspension of the mutant particles. The mutants have been used to investigate the role of individual proteins in ribosome function and assembly. They have also aided in the topographic pinpointing of proteins on the surface of the organelle.

Cloning of nocardioform DNA conferring the ability to inactivate rifampicin.

A novel mechanism of resistance to rifampicin has recently been reported in which this antibiotic is inactivated. Here we describe the cloning of DNA from a nocardiofrom strain conferring this ability to inactivate rifampicin. Cloning was on the basis of complementation, as an increased resistance to the antibiotic. The genetic information was on a 8.3-kb BglII fragment.

Escherichia coli 30S mutants lacking protein S20 are defective in translation initiation.

The 30S ribosomal subunits derived from Escherichia coli TA114, a a temperature-sensitive mutant lacking ribosomal protein S20, were shown to be defective in two ways: (a) they have a reduced capacity for association with the 50S ribosomal subunit which results in the impairment of most of the functions requiring a coordinated interaction between the two subunits; (b) they are defective in functions which do not require their interaction with the large subunit (i.e., the formation of ternary complexes with aminocyl-tRNAs and templates, including the formation of 30S initiation complexes with fMet-tRNA and mRNA). The 30S (-S20) subunits seem to interact normally with both template and aminoacyl-tRNA individually, but appear to be impaired in the rate-limiting isomerization step leading to the formation of a codon-anticodon interaction in the P site.