Cloning and Sequencing of the Spore Germination Gene of Bacillus megaterium ATCC 12872: Similarities to the NaH-Antiporter Gene of Enterococcus hirae

The germination mutant TM-31 of Bacillus megaterium ATCC 12872, was isolated by transposon Tn917 insertional mutagenesis. Glucose, L -proline, L -leucine and KNO₃ germinated TM-31 poorly. The DNA in the region of the Tn917 insertion was cloned, and its nucleotide sequence determined. One major open reading frame was present on the cloned DNA. The hydrophobic protein encoded is presumably membrane-associated. A homology search revealed that the gene encoded in the region of the Tn917 insertion is homologous to napA of Enterococcus hirae. napA codes for the NaH-antiporter. It is hypothesized that transport of cations must play an important role in spore germination in B. megaterium ATCC 12872.     

Distribution and variation of bacitracin synthetase gene sequences in laboratory stock strains of Bacillus licheniformis

Distribution and variation of bacitracin synthetase gene (bac) sequences in 22 laboratory stock strains of Bacillus licheniformis were studied by Southern hybridization of bac gene probes from B. licheniformis ATCC 10716 to genomic PstI or HindIII restriction fragments. Eleven strains gave hybridization signals. These hybridization patterns were classed into two types. Eight strains showed similar patterns to that of ATCC 10716 and all, except one, produced bacitracin. The three strains showed fairly different hybridization patterns from that of ATCC 10716, and one (ATCC 33632) of them produced bacitracin. None of the remaining 11 strains, including ATCC 14580 (type strain), gave any hybridization signals. All strains carrying bac gene sequences were erythromycin resistant. With one exception, all strains without bac gene sequences were erythromycin sensitive. These results show that B. licheniformis strains are divided into two groups with respect to presence of bac gene sequences and erythromycin resistance. Received: 5 September 2001 / Accepted: 19 October 2001     

Genetic Characterization of a New Thermotolerant Bacillus licheniformis Strain

A potentially new thermotolerant B. licheniformis strain (code name I89), producer of an antibiotic active against Gram-positive bacteria, was genetically characterized and compared with the type strain B. licheniformis ATCC 10716, producer of bacitracin. Studies on DNA base composition (G + C content) and DNA reassociation revealed that the two strains show around 76% homology. Nevertheless, results obtained by rRNA hybridization, with a heterologous probe coding for most of the 16S region of the rRNA operon of Bacillus subtilis, revealed differences in the number of copies for that gene and in the hybridization pattern. Additionally, a different restriction digestion pattern was obtained when DNA was digested with the enzymes NotI, SmaI and analyzed by PFGE. The I89 strain holds a 7.6-kb plasmid not present in the reference strain. The existence of various unique restriction sites and also the stability of this plasmid make it ideal for the future development of a cloning and expression vector. Received: 29 June 1999 / Accepted: 1 September 1999     

Construction of a recA mutant of Burkholderia (formerly Pseudomonas), cepacia

A recA mutant was constructed of a soil isolate of Burkholderia cepacia, strain ATCC 17616. Prior to mutagenesis, the recA gene was cloned from this strain by its ability to complement the methyl methanesulfonate sensitivity of an Escherichia coli recA mutant. Sequence analysis of the strain showed high sequence similarity (94% nucleic acid and 99% amino acid identity) with the recA gene previously cloned from a clinical isolate of B. cepacia, strain JN25. The subcloned recA gene from B. cepacia ATCC 17616 restored UV resistance and recombination proficiency to recA mutants of E. coli and Pseudomonas aeruginosa, as well as restoring the ability of D3 prophages to be induced to lytic growth from a RecA⁻ strain of P. aeruginosa. The recA mutant of B. cepacia ATCC 17616 was constructed by λ-mediated Tn5 mutagenesis of the cloned recA gene in E. coli, followed by replacement of the Tn5-interrupted gene for the wild-type allele in the chromosome of B. cepacia by marker exchange. The RecA⁻ phenotype of the mutant was demonstrated by the loss of UV resistance as compared to the parental strain. Southern hybridization analysis of chromosomal DNA from the mutant indicated the presence of Tn5 in the recA gene, and the location of the Tn5 insertion in the recA allele was identified by nucleotide sequence analysis. A test using the recA mutant to see if acquired resistance to d-serine toxicity in B. cepacia might be a result of RecA-mediated activities proved negative; nevertheless, RecA activity potentially contributes to the overall genomic plasticity of B. cepacia and a recA mutant will be useful in bioengineering of this species. Received: 24 January / Received revision: 11 July 1997 / Accepted: 25 August 1997     

Evaluation of the biological containment system based on the Escherichia coli gef gene in Pseudomonas aeruginosa W51D

The cell-killing gef gene was introduced, under the control of the lac promoter and as part of a transposon, into Pseudomonas aeruginosa W51D, a strain able to degrade branched-chain alkylbenzene sulfonates. Only 1% of the cells that inherited the transposon (Tngef) showed a conditional lethal phenotype, and this phenotype was lost at a high frequency without apparent loss of the tetracycline resistance encoded by the transposon. Southern blot analysis of two W51D::Tngef derivatives that expressed the cell-killing function showed multiple insertions of the transposon. These data suggest that Gef protein is able to kill P. aeruginosa W51D, but it seems that the level of resistance to Gef toxin in this stain is higher than that of previously reported bacteria, and that the expression of multiple copies of the gef gene is necessary to attain cell death. The higher level of resistance does not seem to be particular to strain W51D since two other P. aeruginosa strains analyzed (PAO2003 and ATCC 9027) also presented a small proportion of cells expressing the conditional lethal phenotype when they inherited the Tngef transposon. Received: 11 December 1995 / Received revision: 15 July 1996 / Accepted: 5 August 1996     

The transposable elements resident on the plasmids of Pseudomonas putida strain H, Tn 5501 and Tn 5502 , are cryptic transposons of the Tn 3 family

Genes for (methyl)phenol degradation in Pseudomonas putida strain H (phl genes) are located on the plasmid pPGH1. Adjacent to the phl catabolic operon we identified a cryptic transposon, Tn5501, of the Tn3 family (class II transposons). The genes encoding the resolvase and the transposase are transcribed in the same direction, as is common for the Tn501 subfamily. The enzymes encoded by Tn5501, however, show only the overall homology characteristic for resolvases/integrases and transposases of Tn3-type transposons. Therefore it is likely that Tn5501 is not a member of one of the previously defined subfamilies. Inactivation of the conditional lethal sacB gene was used to detect transposition of Tn5501. While screening for transposition events we found another transposon integrated into sacB in one of the sucrose-resistant survivors. This element, Tn5502, is a composite transposon consisting of Tn5501 and an additional DNA fragment. It is flanked by inverted repeats identical to those of Tn5501 and the additional fragment is separated from the Tn5501 portion by an internal repeat (identical to the left terminal repeat). Transposition of phenol degradation genes could not be detected. Analysis of sequence data revealed that the phl genes are not located on a Tn5501-like transposon. Received: 21 July 1997 / Accepted: 7 July 1998     

Mytilus edulis Histone Gene Clusters Containing Only H1 Genes

We isolated five different phage clones containing histone gene clusters with up to five H1 genes per phage clone from a Mytilus edulis genomic library. Among these H1 genes, nine gene types coding for five different H1 proteins have been identified. All H1 histone genes were located on repetitive restriction fragments with only slightly different sizes. The H1 coding regions show highly related sequences, suggesting that the multitude of H1 genes has evolved by gene duplication events. Core histone genes could not be found on these five Mytilus edulis genome fragments. Received: 28 July 1998 / Accepted: 17 May 1999     

Establishment of an Arbitrary PCR for Rapid Identification of Tn917 Insertion Sites in Staphylococcus epidermidis: Characterization of Biofilm-Negative and Nonmucoid Mutants

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.     

Evolutionary relationship of the Bacillus licheniformis macrolide-lincosamide-streptogramin B resistance elements

Summary Naturally occurring erythromycin (Em) resistance was found in 11 of the 18 Bacillus licheniformis isolates tested but was absent from a wide variety of other Bacillus strains. The Em resistance elements confer inducible macrolide-lincosamide-streptogramin B (MLS) resistance and are related to ermD an MLS resistance element previously cloned from the chromosome of B. licheniformis 749. The MLS sensitive B. licheniformis strains and the other sensitive Bacillus strains tested, lack sequences with detectable homology to ermD. The sensitive B. licheniformis strains do exhibit homology to sequences which flank ermD in B. licheniformis 749. The relative sizes of the homologous DNA fragments suggest that the sensitive strains are lacking a 3.6 kb segment which contains ermD. It is shown that ermD is homologous to chromosomal DNA from Streptomyces erythreus ATCC 11635, an Em producing organism. These observations suggest to us that MLS resistance may have arisen in the Streptomyces and spread to B. licheniformis another gram positive bacterium found in soil. It is further proposed that ermD is or was located on a transposon-like element and has spread and evolved further to yeild a variety of related Staphylococcal and Streptococcal MLS determinants.     

Physical mapping of LP51 and LP52 prophages of lysogenic strains of Bacillus licheniformis

Summary The physical maps of the LP51 and LP52 prophages in lysogenic strains of Bacillus licheniformis were constructed on the basis of data obtained by hybridization of phage DNA probes with Southern blots of restricted DNA of the lysogens. The data were compatible with the Campbell model for chromosomal integration; the attP site was mapped at 58.7–61.8 map units of the genomes of both phages. Identification of prophage-host DNA junction fragments indicated the presence of a unique attB site on the bacterial chromosome; the set of junction fragments in the strain B. licheniformis ATCC 10716 was identical to that of ATCC 11946, but different from ATCC 8187. Both the LP51 and LP52 phages used the same integration sites. Upon reinfection with either phage, the cured strains UM12 and UM18 (i.e. 10716 and 11946 cured of LP52 or LP51, respectively) turned out to be integration deficient. In surface cultures the reinfected bacteria could be maintained in the lysogenic state without, however, integrating the phage genome; when these bacteria were passaged in submerged cultures, several modes of anomalous integration were observed, and the phage segregated into a variety of forms, discernible by virulence and plaque morphology. In liquid cultures of UM12(LP51) or UM12(LP52) lytic forms finally predominated, while most lysogenized UM18 were converted into defective lysogens which contained a defective prophage in a stably integrated form.     

Cloning and sequencing of the leu C and npr M genes and a putative spo IV gene from Bacillus megaterium DSM319

The leuC gene, encoding 3-isopropylmalate dehydrogenase, the nprM gene (neutral protease) and a sporulation gene coding for a putative spoIV protein (spoIV) from Bacillus megaterium DSM319 were cloned and the nucleotide sequences were determined. The leuC gene is 1101 bp in length, preceded by a ribosome binding site; no promoter consensus sequence could be found. The nucleotide sequence from nprM when compared to the recently published gene from B. megaterium ATCC 14581 exhibited only a 17-base pair deviation. From a sporulation mutant isolated after transposonmutagenesis with transposon Tn917 the insertion site of the transposon was cloned and adjacent chromosomal fragments were characterized. An open reading frame that encodes for a putative spo protein of 247 amino-acid residues was identified.Sequence data presented in this contribution are part of doctoral theses of the Naturwissenschaftliche Fakultät Münster, Germany nprM (KDW); leuC and spoIV (MB)     

A Corynebacterium glutamicum gene encoding a two-domain protein similar to biotin carboxylases and biotin-carboxyl-carrier proteins

Following the analysis of transposon Tn5432-induced mutants of Corynebacterium glutamicum ATCC 13032, a gene encoding a protein with a biotin-binding motif was cloned. The DNA sequence of this gene revealed an open reading frame encoding 591 amino acids with a calculated mol. mass of 63.4 kDa. The protein is composed of two domains, an N-terminal biotin carboxylase and a C-terminal biotin-carboxyl-carrier protein, that are highly similar to corresponding subunits from prokaryotic and eukaryotic biotin enzymes. Over 70% identity was found to a protein from Mycobacterium leprae proposed to be part of an acyl-CoA carboxylase. Since it was not possible to inactivate the C. glutamicum gene, the gene most likely encodes a subunit of the essential acetyl-CoA carboxylase, which catalyzes the committed step in fatty acid synthesis. Received: 2 December 1995 / Accepted: 20 May 1996     

Bacterial catabolic transposons

The introduction of foreign organic hydrocarbons into the environment in recent years, as in the widespread use of antibiotics, has resulted in the evolution of novel adaptive mechanisms by bacteria for the biodegradation of the organic pollutants. Plasmids have been implicated in the catabolism of many of these complex xenobiotics. The catabolic genes are prone to undergo genetic rearrangement and this is due to their presence on transposons or their association with transposable elements. Most of the catabolic transposons have structural features of the class I (composite) elements. These include transposons for chlorobenzoate (Tn5271), chlorobenzene (Tn5280), the newly discovered benzene catabolic transposon (Tn5542), and transposons encoding halogenated alkanoates and nylon-oligomer-degradative genes. Transposons for the catabolism of toluene (Tn4651, Tn4653, Tn4656) and naphthalene (Tn4655) belong to class II (Tn3 family) elements. Many catabolic genes have been associated with insertion sequences, which suggests that these gene clusters could be rapidly disseminated among the bacterial populations. This greatly expands the substrate range of the microorganisms in the environment and aids the evolution of new and novel degradative pathways. This enhanced metabolic versatility can be exploited for and is believed to play a major part in the bioremediation of polluted environments. Received: 13 July 1998 / Received revision: 22 September 1998 / Accepted: 26 September 1998     

A melanin polyketide synthase (PKS) gene from Nodulisporium sp. that shows homology to the pks1 gene of Colletotrichum lagenarium

The melanin polyketide synthase (pks) gene of Nodulisporium sp. MF5954 (ATCC74245) was cloned by exploiting its homology to the Colletotrichum lagenarium pks1 gene. Sequence analysis demonstrated that this gene is 70% identical to the C. lagenarium pks1 gene. A gene disruption construct, designed to replace both the ketoacyl synthase and acyl transferase domains with a hygromycin resistance (Hy^r) gene, was synthesized, and used to disrupt the Nodulisporium melanin pks1 gene via homologous recombination, resulting in a mel(−) phenotype. Sequence analyses of the gene and of cDNA segments generated by RT-PCR indicate that there are three introns in the 5′ half of the gene. The proposed 2159-amino acid product is 72% identical and 78% similar to the 2187-amino acid sequence deduced from the C. lagenarium pks1 gene. This similarity is notable, considering that C. lagenarium is a member of the order Phyllachoales or Sordariales, whereas Nodulisporium is generally believed to be member of the order Xylariales. However, despite the strong resemblance between the amino acid sequences in the acyl transferase domains of the two proteins, only one in five codons are conserved in the DNA sequences that encode this motif. The Nodulisporium sp. pks1 gene sequence and the amino acid sequence deduced from its coding region have been deposited in Genbank under Accession No. AF151533. Received: 15 May 1999 / Accepted: 26 July 1999     

Metal-induced expressing of mammal Metallothionein-1 gene in cyanobacteria to promote cadmium-binding preferences

The metal(zinc)-inducible smtA gene promoter (smt O-P) from cyanobacteria was applied for the expression of mouse MT-1 cDNA in the filamentous cyanobacterium Anabaena sp. PCC 7120 to enhance its metal-binding capability and to change its main binding specificity from zinc to cadmium. Shuttle expression vector pKT-MRE transformed the cyanobacterial cells by triparent conjugal transfer. Positive clones were screened and identified by streptomycin, DNA dot blot, SDS-PAGE and Western blot analysis. Photosynthetic oxygen evolution and metal atom absorption indicated that under the cadmium stress the metal-induced expression of foreign mMT-1 doubled their cadmium resistance and developed cells showing a much higher preference to absorb cadmium other than zinc in medium. The cadmium content in cell extract rose from 11% to 36%, and the cadmium cleared from media by transgenic cells rose from 18% to 62%. There was only a slight enhancement for zinc binding in the wild or transgenic type. Received: 1 March 1999 / Received last revision: 9 July 1999 / Accepted: 1 August 1999     

The pilA gene of Xanthomonas campestris pv. citri is required for infection by the filamentous phage cf

 Host factors that are important for infection of Xanthomonas campestris pv. citri by the filamentous bacteriophage cf were investigated by transposon mutagenesis with Tn5tac1. A mutant, XT501, that was resistant to cf infection was recovered, showing that the gene inactivated by the transposon is required for infection by the phage but not for cf replication or assembly. A 1.7-kb SacI-ApaI DNA fragment from XT501 containing the bacterial DNA flanking one end of the transposon was cloned and shown to be required for cf infection. Nucleotide sequence analysis of the 1.7-kb fragment reveals the presence of an ORF that encodes a protein of 146 amino acids. This protein shows 42% identity to the type 4 prepilin encoded by the pilA genes of other bacteria. The pilA gene of X. campestris pv. citri is thus essential for infection by the bacteriophage cf. Received: 30 November 1998 / Accepted: 21 April 1999     

Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene

Summary A 37.5 kb region encompassing a set of the naphthalene degrading genes on the Pseudomonas plasmid NAH7 was found to be transposable only in the presence of the transposase encoded by the Tn1721 subgroup of the class II transposons. This newly identified mobile element, designated Tn4655, contained short (38 bp) terminal inverted repeats which shared extensive sequence homology with those of members of the Tn1721 subgroup. Tn4655 transposed by a two-step process involving formation of the cointegrate followed by its subsequent resolution. In contrast to the defect in the trans-acting factor for the first step, a functional system for the latter step was encoded within a 2.4 kb region in Tn4655. Analysis of deletion and insertion mutants demonstrated that the 2.4 kb region contained the cis-acting (res) site and the gene for a trans-acting factor (resolvase); complementation analysis indicated that Tn4655 resolvase function was not interchangeable with those of other well-studied class 11 transposons, including the Tn1721 subgroup. Tn4655 had no DNA sequences that were hybridizable with the transposase or resolvase genes of the Tn1721 subgroup.Abbreviations Ap ampicillin - Cb carbenicillin - Cm chloramphenicol - Km kanamycin - Sm streptomycin - Tc tetracycline - Tp trimethoprim     

Construction of a Slime Negative Transposon Mutant in Staphylococcus epidermidis Using the Enterococcus faecalis Transposon Tn917

The slime-producing Staphylococcus epidermidis strain sensu strictu CNS23 was transformed by protoplast transformation with the plasmid pTV1 which carries transposon Tn917. Using this transposon mutagenesis system we obtained the Tn917-inserted mutant CT512, which has lost the ability to produce slime. A single insertion of the trasposon Tn917 into the chromosome of CT512 could be detected by Southern hybridization. This mutant showed a significantly higher stability concerning its slime-negative phenotype compared with spontaneous slime-negative mutants of S. epidermidis strain CNS23. In slime-ELISA no slime-associated antigen could be detected in extracts of the transposon mutant. Compared to slime-positive S. epidermidis strains, CT512 lacked in accumulative growth in microtiter tube test.     

Resolution of a hybrid cointegrate between transposons Tn501 and Tn1721 defines the recombination site

Summary The related transposons Tn501 and Tn1721 have a 3.8 kb region in common that contains two genes (tnpA and tnpR) and a resolution site (res) required for transposition. Resolvase, the product of tnpR, catalyses site-specific recombination at res, a 186 base pair (bp) sequence located adjacent to tnpR at one end of the homology region. We describe here identification of the crossover site within res. It involved the construction of a plasmid containing copies of res (Tn501) and res (Tn1721) in direct orientation and tnpR-mediated intramolecular recombination between the two homologous (but non-identical) sites. The resulting hybrid contained Tn501 and Tn1721 fused at the crossover point. DNA sequence analysis of the recombinant indicates that recombination occurs in an 11 bp region of exact homology between Tn501 and Tn1721. The recombination site lies 161–172 bp upstream of tnpR at the transition from homology to non-homology between Tn501 and Tn1721 suggesting that site-specific recombination may have played a role in the evolution of these elements.