Identification and characterization of the regulatory elements of the inducible acetamidase operon from Mycobacterium smegmatis

The highly inducible acetamidase promoter from Mycobacterium smegmatis has been used as a tool in the study of mycobacterial genetics. The 4.2 kb acetamidase operon contains four putative open reading frames (ORFs) (amiC, amiA, amiD, and amiS) upstream of the 1.2 kb acetamidase ORF (amiE). In this article, using electrophoretic mobility shift assay and promoter probe analyses with a lacZ reporter system, we show the position of three putative operators within the acetamidase operon in M. smegmatis. Results from these studies reinforce previous findings about the involvement of multiple promoters in the regulation of acetamidase gene expression. Each of the identified operators are positioned upstream of the respective promoter reported in previous studies. We also found that the crude cell lysate of M. smegmatis containing potential regulators, obtained from bacteria grown under inducing or noninducing conditions, binds to specific operators. The binding affinity of each operator with its cognate regulator is significantly different from the other. This supports not only the previous model of acetamidase gene regulation in M. smegmatis but also explains the role of these operators in controlling the expression of respective promoters under different growth conditions.     

Molecular characterization of AmiC,a positive regulator in acetamidase operon of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

Mycobacterium smegmatis, a rapidly growing non-pathogenic mycobacterium, is currently used as a model organism to study mycobacterial genetics. Acetamidase of M. smegmatis is the highly inducible enzyme of Mycobacteria, which utilizes several amide compounds as sole carbon and nitrogen sources. The acetamidase operon has a complex regulatory mechanism, which involves three regulatory proteins, four promoters, and three operator elements. In our previous study, we showed that over-expression of AmiA leads to a negative regulation of acetamidase by blocking the P2 promoter. In this study, we have identified a new positive regulatory protein, AmiC that interacts with AmiA through protein-protein interaction. Gel mobility shift assay showed that AmiC protein inhibits AmiA from binding to the P2 promoter. Interaction of AmiC with cis-acting elements identified its binding ability to multiple regulatory regions of the operon such as P3, OP3, and P1 promoter/operator. Consequently, the addition of inducer acetamide to AmiC complexe trips the complexes, causing AmiC to appear to be the sensory protein for the amides. Homology modeling and molecular docking studies suggest AmiC as a member of Periplasmic binding proteins, which preferentially bind to the inducers and not to the suppressor. Over-expression of AmiC leads to down-regulation of the negative regulator, amiA, and constitutive up-regulation of acetamidase. Based on these findings, we conclude that AmiC positively regulates the acetamidase operon.     

Specific Structural Features of the N-Acetylmuramoyl-l-Alanine Amidase AmiD from Escherichia coli and Mechanistic Implications for Enzymes of This Family

AmiD is the fifth identified N-acetylmuramoyl-l-alanine zinc amidase of Escherichia coli. This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of cleaving the intact peptidoglycan (PG) as well as soluble fragments containing N-acetylmuramic acid regardless of the presence of an anhydro form or not, unlike the four other amidases, AmiA, AmiB, AmiC, and AmpD, which have some specificity. AmiD function is, however, not clearly established but it could be part of the enzymatic machinery involved in the PG turnover in E. coli. We solved three structures of the E. coli zinc amidase AmiD devoid of its lipidic anchorage: the holoenzyme, the apoenzyme in complex with the substrate anhydro-N-acetylmuramic-acid-l-Ala-γ-d-Glu-l-Lys, and the holoenzyme in complex with the l-Ala-γ-d-Glu-l-Lys peptide, the product of the hydrolysis of this substrate by AmiD. The AmiD structure shows a relatively flexible N-terminal extension that allows an easy reach of the PG by the enzyme inserted into the outer membrane. The C-terminal domain provides a potential extended geometrical complementarity to the substrate. AmiD shares a common fold with AmpD, the bacteriophage T7 lysozyme, and the PG recognition proteins, which are receptor proteins involved in the innate immune responses of a wide range of organisms. Analysis of the different structures reveals the similarity between the catalytic mechanism of zinc amidases of the AmiD family and the thermolysin-related zinc peptidases.     

Expression,purification and functional characterization of AmiA of acetamidase operon of Mycobacterium smegmatis

Regulation of gene expression is one of the mechanisms of virulence in pathogenic organisms. In this context, we would like to understand the gene regulation of acetamidase enzyme of Mycobacterium smegmatis, which is the first reported inducible enzyme in mycobacteria. The acetamidase is highly inducible and the expression of this enzyme is increased 100-fold when the substrate acetamide is added. The acetamidase structural gene (amiE) is found immediately downstream of three predicted open reading frames (ORFs). Three of these genes along with a divergently expressed ORF are predicted to form an operon and involved in the regulation of acetamidase enzyme. Here we report expression, purification and functional characterization of AmiA which is one of these predicted ORFs. Electrophoretic mobility shift assays showed that AmiA binds to the region between the amiA and amiD near the predicted promoter (P2). Over-expression of AmiA significantly lowered the expression of acetamidase compared to the wild type as demonstrated by qRT-PCR and SDS-PAGE. We conclude that AmiA binds near P2 promoter and acts as a repressor in the regulation of acetamidase operon. The described work is a further step forward toward broadening the knowledge on understanding of the complex gene regulatory mechanism of Mycobacterium sp.     

Isolation and phenotypic characterization of Pseudomonas aeruginosa pseudorevertants containing suppressors of the catabolite repression control-defective crc-10 allele

The amiE gene encodes an aliphatic amidase capable of converting fluoroacetamide to the toxic compound fluoroacetate and is one of many genes whose expression is subject to catabolite repression control in Pseudomonas aeruginosa. The protein product of the crc gene, Crc, is required for repression of amiE and most other genes subject to catabolite repression control in this bacterium. When grown in a carbon source such as succinate, wild-type P. aeruginosa is insensitive to fluoroacetamide (due to repression of amiE expression). In contrast, mutants harboring the crc-10 null allele cannot grow in the presence of fluoroacetamide (due to lack of repression of amiE). Selection for succinate-dependent, fluoroacetamide-resistant derivatives of the crc-10 mutant yielded three independent pseudorevertants containing suppressors that restored a degree of catabolite repression control. Synthesis of Crc protein was not reestablished in these pseudorevertants. All three suppressors of crc-10 were extragenic, and all three also suppressed a Delta crc::tetA allele. In each of the three pseudorevertants, catabolite repression control of amidase expression was restored. Catabolite repression control of mannitol dehydrogenase production was also restored in two of the three isolates. None of the suppressors restored repression of glucose-6-phosphate dehydrogenase or pyocyanin production.     

A mutation, adjacent to gene amdS, defining the site of action of positive-control gene amdR in Aspergillus nidulans

In Aspergillus nidulans the acetamidase enzyme is inducible by omega-amino acids, sources of acetyl-coenzyme A, and benzoate. The amdR (or intA) gene is a positive-control gene involved in omega-amino acid induction only. A cis-acting mutation amdI93 located in a complex controlling region adjacent to the acetamidase structural gene was found to abolish induction by omega-amino acids but not induction by other sources of induction. As predicted, this mutation was epistatic to constitutive amdR alleles but did not affect the expression of mutations in other regulatory genes.     

Cloning of the wide spectrum amidase gene from Brevibacterium sp. R312 by genetic complementation. Overexpression in Brevibacterium sp. and Escherichia coli

Abstract The amiE gene of Brevibacterium sp. R312 encoding wide spectrum amidase was isolated by complementation of a Brevibacterium sp. mutant using a plasmid gene bank of chromosomal DNA. The amiE structural gene and its promoter were localized on a 1.8-kb fragment by subsequent subcloning and complementation studies. In Brevibacterium sp., the investigation of amidase activities related to one copy of the gene suggested that the regulation of the amiE gene expression was under negative control. High expression levels have been obtained in Brevibacterium sp. and, after substitution of the amiE promoter by the tac promoter, in Escherichia coli .     

Instability of the acetamide-inducible expression vector pJAM2 in Mycobacterium tuberculosis

The Escherichia coli-mycobacterium shuttle vector pJAM2 has been used to inducibly express genes in mycobacteria. The vector carries the promoter region from the highly inducible acetamidase gene of Mycobacterium smegmatis which is used to drive expression of heterologous genes. We used pJAM2 to over-express the Mycobacterium tuberculosis gene Rv2868c, a homologue of gcpE. In M. smegmatis the plasmid was stable, but the promoter region was readily deleted when the parental vector or recombinant plasmids were transformed into M. tuberculosis. We mapped the deletion by sequencing and found that it encompassed the entire acetamidase promoter and adjacent sequence totalling approximately 7.3 kb and occurred very soon after introduction into M. tuberculosis. This is the first report of instability of a vector carrying the acetamidase promoter in M. tuberculosis.     

Identification and structure of the nasR gene encoding a nitrate- and nitrite-responsive positive regulator of nasFEDCBA (nitrate assimilation) operon expression in Klebsiella pneumoniae M5al.

Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources through the nitrate assimilatory pathway. The structural genes for assimilatory nitrate and nitrite reductases together with genes necessary for nitrate transport form an operon, nasFEDCBA. Expression of the nasF operon is regulated both by general nitrogen control and also by nitrate or nitrite induction. We have identified a gene, nasR, that is necessary for nitrate and nitrite induction. The nasR gene, located immediately upstream of the nasFEDCBA operon, encodes a 44-kDa protein. The NasR protein shares carboxyl-terminal sequence similarity with the AmiR protein of Pseudomonas aeruginosa, the positive regulator of amiE (aliphatic amidase) gene expression. In addition, we present evidence that the nasF operon is not autogenously regulated.     

Development of inducible systems to engineer conditional mutants of essential genes of Helicobacter pylori

The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacI(q)-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacI(q) repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring beta-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-beta-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.     

Induction of the acetamidase of Aspergillus nidulans by acetate metabolism.

Growth tests and enzyme determinations strongly suggest that the acetamidase of Aspergillus nidulans is induced by a product of acetate metabolism rather than the substrate, acetamide. The cis-dominant mutation, amdI9, which is closely linked to amdS, the structural gene for the acetamidase, results in greatly increased sensitivity to induction by acetate metabolism. Propionate, L-threonine, and ethanol also result in acetamidase induction. Mutations in the facA, facB, and facC genes, which lead to low levels of acetyl-coenzyme A synthase, are epistatic to the amdI9 mutation for strong growth on acetamide medium and abolish acetamide and propionamide induction of the acetamidase and isocitrate lyase enzymes. Acetate, L-threonine, and ethanol, however, can induce these enzymes in strains containing facA and facC lesions but not in strains containing a facB lesion. The evidence suggests that acetamidase and isocitrate lyase may be induced by a similar mechanism.     

A versatile vector for mycobacterial protein production with a functional minimized acetamidase regulon

Recombinant protein expression is a prerequisite for diverse investigations of proteins at the molecular level. For targets from Mycobacterium tuberculosis it is favorable to use M. smegmatis as an expression host, a species from the same genus. In the respective shuttle vectors, target gene expression is controlled by the complex tetra‐cistronic acetamidase regulon. As a result, the size of those vectors is large, rendering them of limited use, especially when the target proteins are expressed from multi‐cistronic operons. Therefore, in the current work we present a versatile new expression vector in which the acetamidase regulon has been minimized by deleting the two genes amiD and amiS. We assessed the functional properties of the resulting vector pMyCA and compared it with those of the existing vector pMyNT that contains the full‐length acetamidase regulon. We analyzed the growth features and protein expression patterns of M. smegmatis cultures transformed with both vectors. In addition, we created mCherry expression constructs to spectroscopically monitor the expression properties of both vectors. Our experiments showed that the minimized vector exhibited several advantages over the pMyNT vector. First, the overall yield of expressed protein is higher due to the higher yield of bacterial mass. Second, the heterologous expression was regulated more tightly, offering an expression tool for diverse target proteins. Third, it is suitable for large multi‐protein complexes that are expressed from multi‐cistronic operons. Additionally, our results propose a new understanding of the regulation mechanism of the acetamidase regulon with the potential to construct more optimized vectors in the future.     

An anhydro-N-acetylmuramyl-L-alanine amidase with broad specificity tethered to the outer membrane of Escherichia coli

From its amino acid sequence homology with AmpD, we recognized YbjR, now renamed AmiD, as a possible second 1,6-anhydro-N-acetylmuramic acid (anhMurNAc)-l-alanine amidase in Escherichia coli. We have now confirmed that AmiD is an anhMurNAc-l-Ala amidase and demonstrated that AmpD and AmiD are the only enzymes present in E. coli that are able to cleave the anhMurNAc-l-Ala bond. The activity was present only in the outer membrane fraction obtained from an ampD mutant. In contrast to AmpD, which is specific for the anhMurNAc-l-alanine bond, AmiD also cleaved the bond between MurNAc and l-alanine in both muropeptides and murein sacculi. Unlike the periplasmic murein amidases, AmiD did not participate in cell separation. ampG mutants, which are unable to import GlcNAc-anhMurNAc-peptides into the cytoplasm, released mainly peptides into the medium due to AmiD activity, whereas an ampG amiD double mutant released a large amount of intact GlcNAc-anhMurNAc-peptides into the medium.     

Mutants with Altered Glucose Repression of Amidase Enzymes in Aspergillus nidulans

Aspergillus nidulans produces acetamidase and formamidase enzymes. The acetamidase is produced in reduced amounts during growth on glucose, whereas the formamidase is not greatly affected. Mutations in a gene, amdT, which affect glucose repression of amidases are described. One of these, amdT102, causes the acetamidase to be no longer subject to glucose repression and also affects ability to synthesize formamidase. The other, amdT19, results in both the formamidase and the acetamidase being subject to abnormally strong glucose repression, and also in increased maximal acetamidase activities. The dominance relationships at the amdT locus have been investigated. It is suggested that the amdT gene may play a positive role in controlling amidase synthesis.     

Transformation by integration in Aspergillus nidulans

DNA-mediated genetic transformation of Aspergillus nidulans has been achieved by incubating protoplasts from a strain of A. nidulans carrying a deletion in the acetamidase structural gene with DNA of derivatives of plasmid pBR322 containing the cloned structural gene for acetamidase [Hynes et al., Mol. Cell. Biol. 3 (1983) 1430-1439; p3SR2] in the presence of polyethylene glycol and CaCl2. The highest frequency obtained was 25 transformants per microgram of DNA. No enhancement of the transformation frequency was observed when DNAs of plasmids carrying either a fragment of the A. nidulans ribosomal repeat (p3SR2rr) or a fragment containing a possible A. nidulans mitochondrial origin of replication (p3SR2mo) in addition to the acetamidase gene were used. Both pBR322 and acetamidase gene sequences become integrated into the genome of A. nidulans in transformant strains. Integration events into the residual sequences adjacent to the deletion in the acetamidase gene, and probably (for p3SR2rr and p3SR2mo) into the ribosomal repeat unit are described.