Cloning the alpha-amylase gene of Bacillus amyloliquefaciens in cyanobacteria cells

The recombinant plasmids of pIAH4amy series were constructed containing the alpha-amylase gene of Bacillus amyloliquefaciens A50 with its own promoter and leading sequence within an integrative vector plasmid pIAH4 (CmR) for cyanobacterium Anacystis nidulans R2. At Anacystis nidulans transformation the hybrid plasmids integrate into cyanobacterium chromosome with high efficiency and all CmR transformants produce alpha-amylase. Expression of bacillar alpha-amylase gene in cyanobacterium cells is independent of the cloned gene orientation in the vector plasmid. Secretion of alpha-amylase into the cyanobacterial periplasm has been demonstrated.     

A new approach for molecular cloning in cyanobacteria: cloning of an Anacystis nidulans met gene using a Tn901-induced mutant

A new strategy for molecular cloning in the cyanobacterium Anacystis nidulans R-2 is described. This strategy involved the use of a transposon and was developed for the cloning of a gene encoding methionine biosynthesis. A met::Tn901 mutant was isolated. Chromosomal DNA fragments were cloned in the Escherichia coli plasmid vector pACYC184. A recombinant plasmid carrying the inactivated met::Tn901 gene was selected after transformation to E. coli. The cloned met::Tn901 DNA fragment was used as a probe to select the corresponding A. nidulans R-2 wild-type met gene from a gene library prepared in E. coli, using the newly constructed shuttle cosmid vector pPUC29. When transformed into A. nidulans Met- mutants, this cloned gene allowed the mutants to grow prototrophically.     

Instability of Tn5 in a plasmid cloning vector for the cyanobacterium Anacystis nidulans.

Transposon Tn5 was used to produce insertions within the region of a cyanobacterial shuttle vector previously identified as necessary for transformation of Anacystis nidulans. These transposon-containing plasmids were used to transform a plasmid-cured derivative of Anacystis strain R2 and tested for structural stability of the transforming plasmid. The transposon DNA was deleted from all the plasmids containing Tn5 within the cyanobacterial replication region. Inserts in the vector DNA were physically stable and expressed the kanr gene. The internal Tn5 HindIII fragment was also cloned into each of the three HindIII sites in the shuttle plasmid. Inserts in two of these sites were stable, whereas inserts into the third site were not.     

Organization of the genes for protein synthesis elongation factors Tu and G in the cyanobacterium Anacystis nidulans.

The genes for protein synthesis elongation factors Tu and G were cloned from the cyanobacterium Anacystis nidulans. The locations of these genes were mapped within the cloned DNA fragment by hybridization with Escherichia coli probes. The organization of the cloned fragment and the DNA flanking it in the A. nidulans chromosome was also determined. The elongation factor Tu and G genes are adjacent to one another and in the same 5'-to-3' orientation. In contrast to other gram-negative bacteria, A. nidulans contains only one gene for elongation factor Tu.     

Recombinant plasmids capable of integration into the chromosome of the cyanobacterium Anacystis nidulans R2

Recombinant plasmids, series pIAB and pIAH, have been constructed by insertion of BamHI or HindIII chromosomal fragments from Anacystis nidulans R2 into the tet gene of plasmid pACYC184. Plasmids pIAB and pIAH are stably maintained in Escherichia coli cells and transfer the CmR marker in transformation of Anacystis nidulans. Blot hybridization technique has shown the formation of CmR clones in transformation to result from integration of plasmid pACYC184 with the chromosome of cyanobacterium.     

Self-cloning in the cyanobacterium Anacystis nidulans R2: Fate of a cloned gene after reintroduction

Functional analysis of cloned genes often makes use of complementation after introducing these genes into cells of a mutant strain. Problems with this self-cloning step in the cyanobacterium Anacystis nidulans R2 have been encountered, which were mainly due to recombinational instability of gene and vector after transformation. Therefore, conditions determining the exchange of material between chromosome, insert and plasmids were studied to achieve the necessary stability. The fate of plasmid pME1, containing a wild-type methionine gene from A. nidulans R2, was investigated after its introduction into a Tn901-induced methionine mutant strain as recipient, so that the mutant chromosomal gene could be distinguished from the plasmid-borne wild-type copy. Two different recipients were constructed, one containing and one lacking the resident plasmid pCH1, which is a derivative of the indigenous small plasmid pUH24. When using the pCH1-free strain and with combined selection for both wild-type gene and vector, the original configuration of the genes in chromosome and vector was retained in the majority of the transformed cells, while the remaining transformants were reciprocal recombinants; under conditions of single selection mainly nonreciprocal recombination or loss of the vector was observed. When the recipient strain contained pCH1 additional recombinational events took place. The results show that under appropriate conditions a chromosomal gene cloned on a plasmid vector can be stably maintained in a majority of the transformants, thus making self-cloning experiments feasible in A. nidulans R2. On the other hand, the introduction of foreign DNA into the chromosome can be achieved by deliberately exploiting recombination between chromosome and plasmid.     

Hybrid vectors for the cyanobacterium Anacystis nidulans R2, containing the plasmid pUB110 from Staphylococcus aureus

The recombinant vector plasmids were constructed having the DNA of pUB110 plasmid (4,5 kb, KmR) from Staphylococcus aureus inserted into the cryptic plasmids pANS (8 Kb) and pANL (48,5 kb) of cyanobacterium Anacystis nidulans R2. The hybrid plasmids transform cyanobacterial cells to Km-resistance with high efficiency. The plasmid pBS20, containing the complete sequence of pANS and pUB110 DNA, transforms Bacillus subtilis rec E4 protoplasts being, however, unstable in bacilli cells and disintegrates deriving a parent pUB110 plasmid.     

Overexpression and sequence of the Escherichia coli cheY gene and biochemical activities of the CheY protein

Anacystis nidulans, a non-nitrogen-fixing cyanobacterium, can fulfill its nitrogen requirement by the assimilation of nitrate. The first step in the pathway, the reduction of nitrate to nitrite, is catalyzed by the molybdo-protein nitrate reductase. In this study, newly developed techniques for gene cloning in A. nidulans R2 were used for the isolation of two genes involved in nitrate reduction. One gene was cloned by complementation of the corresponding mutant; the other gene was picked up from a cosmid gene library by using a restriction fragment containing the transposon-inactivated gene as a probe. Both genes were unlinked single-copy chromosomal genes. Transformation studies provided evidence for the existence of a third locus involved in nitrate reduction.     

Cloning of a third nitrate reductase gene from the cyanobacterium Anacystis nidulans R2 using a shuttle cosmid library

A strategy for gene cloning in the cyanobacterium Anacystis nidulans R2 was developed which made use of a gene library constructed in a shuttle cosmid vector. The method involved phenotypic complementation of mutants with pooled cosmid DNA. The development of the procedure and its application to the cloning of a third gene involved in nitrate reduction are described.     

Shuttle cloning vectors for the cyanobacterium Anacystis nidulans.

Hybrid plasmids capable of acting as shuttle cloning vectors in Escherichia coli and the cyanobacterium Anacystis nidulans R2 were constructed by in vitro ligation. DNA from the small endogenous plasmid of A. nidulans was combined with two E. coli vectors, pBR325 and pDPL13, to create vectors containing either two selectable antibiotic resistance markers or a single marker linked to a flexible multisite polylinker. Nonessential DNA was deleted from the polylinker containing plasmid pPLAN B2 to produce a small shuttle vector carrying part of the polylinker (pCB4). The two polylinker-containing shuttle vectors, pPLAN B2 and pCB4, transform both E. coli and A. nidulans efficiently and provide seven and five unique restriction enzyme sites, respectively, for the insertion of a variety of DNA fragments. The hybrid plasmid derived from pBR325 (pECAN1) also transforms both E. coli and A. nidulans, although at a lower frequency, and contains two unique restriction enzyme sites.     

Thioredoxin is essential for photosynthetic growth. The thioredoxin m gene of Anacystis nidulans

We have taken advantage of the transformation properties of the cyanobacterium Anacystis nidulans R2 to investigate the importance of thioredoxin for photosynthetic growth. The gene encoding thioredoxin m, designated trxM, was cloned from A. nidulans using a synthetic oligonucleotide probe. Based on the nucleotide sequence, thioredoxin m of A. nidulans is composed of 107 amino acids and shares 84, 48, and 48% sequence identity with thioredoxins from Anabaena, spinach, and Escherichia coli, respectively. The trxM gene is single copy and is transcribed on a 510-nucleotide mRNA. We demonstrate that disruption of the trxM gene with a kanamycin resistance gene cartridge is a lethal mutation. Although dispensable in E. coli, thioredoxin is essential for the photosynthetic growth of A. nidulans.     

Isolation of the Rhodopseudomonas sphaeroides form I ribulose 1,5-bisphosphate carboxylase/oxygenase large and small subunit genes and expression of the active hexadecameric enzyme in Escherichia coli

A library of cloned Rhodopseudomonas sphaeroides DNA was screened by colony hybridization for form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) sequences using heterologous RuBPC/O probes. A recombinant plasmid was identified that hybridized to both the Anacystis nidulans and the R. sphaeroides form II RuBPC/O genes. Subcloning of a hybridizing 4-kb SmaI fragment allowed expression of active enzyme in Escherichia coli that was identical to form I RuBPC/O based on polyacrylamide gel electrophoresis and Western immunoblot analysis.     

The gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is located close to the gene for the large subunit in the cyanobacterium Anacystis nidulans 6301.

The gene for the small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase from a cyanobacterium, Anacystis nidulans 6301, has been cloned and subjected to sequence analysis. The SS coding region is located close to and downstream from the large subunit (LS) coding region on the same DNA strand. The spacer region between the LS and the SS coding regions contains 93 base pairs (bp), and has no promoter-like sequences. The coding region of A. nidulans SS gene contains 333 bp (111 codons). The deduced amino acid sequence of the A. nidulans SS protein shows 40% homology with those of higher plants.     

Construction of a hybrid plasmid capable of replication in the bacterium Escherichia coli and the cyanobacterium Anacystis nidulans.

A hybrid plasmid was constructed between the 5.3-megadalton plasmid (pUH24) of Anacystis nidulans R2 and the Escherichia coli plasmid pBR322. This was accomplished by adding a transposon to pBR322 and transforming this DNA into A. nidulans. One resultant hybrid, pLS103, had a molecular weight of 6.8 x 10(6), replicated in both organisms, had unique sites for two restriction endonucleases, conferred ampicillin resistance on both organisms, and could be used as a cloning vector in A. nidulans.     

Cloning and characterization of a photolyase gene from the cyanobacterium Anacystis nidulans.

A 2 kb fragment was isolated from an Anacystis nidulans genomic DNA library by hybridization with synthetic oligonucleotide probes derived from the N-terminal amino acid sequence of Anacystis photolyase. This fragment contains a 1452 bp-long open reading frame encoding a polypeptide of 484 amino acids (Mr 54475). Antibodies raised against purified Anacystis photolyase reacted with extracts of cells harboring fused genes between lacZ of Escherichia coli and this gene. A 40.7% similarity was found between the deduced amino acid sequences of Anacystis and E. coli photolyases, notwithstanding the difference in chromophore structure.     

A hybrid plasmid is a stable cloning vector for the cyanobacterium Anacystis nidulans R2.

Anacystis nidulans R2 is a highly transformable strain which is suitable as a recipient for molecular cloning in cyanobacteria. In an effort to produce an appropriate cloning vector, we constructed a hybrid plasmid molecule, pSG111, which contained pBR328 from Escherichia coli and the native pUH24 plasmid of A. nidulans. pSG111 replicated in and conferred ampicillin and chloramphenicol resistance to both hosts. It contained unique sites for the restriction enzymes EcoRI, SalI, SphI, and XhoI, which could be used for the insertion of exogenous DNA. To demonstrate that a molecule like pSG111 could serve as a shuttle vector for the cloning of A. nidulans genes, we constructed a hybrid plasmid, pRNA404, containing an A. nidulans rRNA operon. This recombinant molecule was genetically and structurally stable during passage through A. nidulans and E. coli. The stability of the hybrid plasmid and the inserted rRNA operon demonstrates the feasibility of cloning in A. nidulans with hybrid vectors, with the subsequent retrieval of cloned sequences.     

phrA, the major photoreactivating factor in the cyanobacterium Synechocystis sp. strain PCC 6803 codes for a cyclobutane-pyrimidine-dimer-specific DNA photolyase

A new broad-host-range plasmid, pSL1211, was constructed for the over-expression of genes in Synechocystis sp. strain PCC 6803. The plasmid was derived from RSF1010 and an Escherichia coli over-expression plasmid, pTrcHisC. Over-expressed protein is made with a removable N-terminal histidine tag. The plasmid was used to over-express the phrA gene and purify the gene product from Synechocystis sp. strain PCC 6803. PhrA is the major ultraviolet-light-resistant factor in the cyanobacterium. The purified PhrA protein exhibited an optical absorption spectrum similar to that of the cyclobutane pyrimidine dimer (CPD) DNA photolyase from Synechocuccus sp. strain PCC 6301 (Anacystis nidulans). Mass spectrometry analysis of PhrA indicated that the protein contains 8-hydroxy-5-deazariboflavin and flavin adenine dinucleotide (FADH2) as cofactors. PhrA repairs only cyclobutane pyrimidine dimer but not pyrimidine (6-4) pyrimidinone photoproducts. On the basis of these results, the PhrA protein is classified as a class I, HDF-type, CPD DNA photolyase.