Plasmid encoded antibiotics inhibit protozoan predation of Escherichia coli K12.

Bacterial plasmids and phages encode the synthesis of toxic molecules that inhibit protozoan predation. One such toxic molecule is violacein, a purple pigmented, anti-tumour antibiotic produced by the Gram-negative soil bacterium Chromobacterium violaceum. In the current experiments a range of Escherichia coli K12 strains were genetically engineered to produce violacein and a number of its coloured, biosynthetic intermediates. A bactivorous predatory protozoan isolate, Colpoda sp.A4, was isolated from soil and tested for its ability to 'graze' on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan "plaque" formation. Using this assay, E. coli K12 strains producing violacein were highly resistant to protozoan predation. However E. coli K12 strains producing violacein intermediates, showed low or no resistance to predation. In separate experiments, when either erythromycin or pentachlorophenol were added to the plaque assay medium, protozoan predation of E. coli K12 was markedly reduced. The inhibitory effects of these two molecules were removed if E. coli K12 strains were genetically engineered to inactivate the toxic molecules. In the case of erythromycin, the E. coli K12 assay strain was engineered to produce an erythromycin inactivating esterase, PlpA. For pentachlorophenol, the E. coli K12 assay strain was engineered to produce a PCP inactivating enzyme pentachlorophenol-4-monooxygenase (PcpB). This study indicates that in environments containing large numbers of protozoa, bacteria which use efflux pumps to remove toxins unchanged from the cell may have an evolutionary advantage over bacteria which enzymatically inactivate toxins.

Antibiotic resistant mutants of Escherichia coli K12 show increases in heterologous gene expression.

Using a variety of antibiotics, it was found that nine separate isolates of spontaneous antibiotic resistant mutants of Escherichia coli K12 pPSX-vioABCDE overproduce the anti-tumour antibiotic violacein. Subsequent analysis showed that seven of these mutations occurred on the plasmid pPSX-vioABCDE. The other two overproducing strains carried spontaneous chromosomal mutations to lincomycin and kanamycin. The kanamycin resistant mutant of E. coli K12 DH10B (AA23) and a lincomycin resistant mutant of E. coli K12 LE392 (AA24) increased the synthesis of violacein. The plasmid pPSX-vioABCDE opv-1 contains a violacein over-production (opv-1) mutation which when introduced into either E. coli K12 AA23 or AA24, resulted in a hyper-production of violacein. Remarkably, E. coli K12 AA23 pPSX-vioABCDE opv-1 produced 41 times the normal level of violacein. In addition, both E. coli K12 AA23 and E. coli K12 AA24 demonstrated an increase in expression of an alpha amylase gene from Streptomyces lividans and the urease gene cluster from Klebsiella oxytoca. These results suggest that selection of antibiotic resistant mutants can increase heterologous gene expression in E. coli K12. Additionally, the increased expression is a general effect applicable to genes and gene clusters cloned into E. coli K12 from both Gram-positive and Gram-negative bacteria.

Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12.

Previous studies showed that when pPSX-vioABCDE was used to transform E. coli K12 DH5alpha the strain retained the plasmid even after 100 generations of unselected growth but produced a low level of the anti-tumour antibiotic violacein. Markedly higher levels of violacein synthesis were obtained from E. coli K12 DH5alpha pUC18-vioABCDE and Sphingomonas sp. JMP4092 pPSX-vioABCDE. Unfortunately, both strains were extremely unstable regardless of presence or absence of antibiotic selection to retain the plasmid. The current study was undertaken to determine if strains of E. coli K12 could be isolated which stably over produce violacein. When a range of E. coli K12 strains were transformed with pPSX-vioABCDE, most produced small amounts of violacein. However, a small number of related strains of E. coli K12 JM101, JM105 and JM109 not only over-produced violacein, but also maintained the high stability. In addition, E. coli K12 JM109 strongly expressed an alpha amylase gene (amyA) from Streptomyces lividans indicating that the S. lividans amyA promoter is highly active in E. coli K12 JM109. In another set of experiments, a violacein overproduction mutation (opv-1) of the plasmid pPSX-vioABCDE was isolated which enabled E. coli K12 DH5alpha to overproduce violacein while retaining high stability. The plasmid pPSX-vioABCDEopv-1 possesses a single base pair deletion in the promoter region of the violacein operon. By combining the over producing strain E. coli K12 JM109 and the over producing plasmid pPSX-vioABCDEopv-1, a stable hyper producing strain (E. coli K12 JM109 pPSX-vioABCDEopv-1) was constructed. Finally, two additional stable vectors, pPSX10 and pPSX20, were constructed to facilitate subcloning and functional analysis studies.

Complete sequence and analysis of the stability functions of pPSX, a vector that allows stable cloning and expression of Streptomycete genes in Escherichia coli K12.

The broad host range, cloning and expression vector pPSX has been completely sequenced and analysed. pPSX is 14.7kb in length and contains the fusion of two continuous segments of the parental 34kb, IncW plasmid pR388. pPSX appears to have retained at least three sets of gene/s which contribute in different ways to plasmid stability. The first of these parB, is a known participant in the partitioning of low-copy number plasmids. While the adjoining gene, orf35, has high homology with kfrA, a putative plasmid nucleoid organiser that is often associated with the ParAB family of proteins. The second set of genes; orfs18, 19, 20, whose exact functions are not clear, have homology to the stability operons of both IncW and IncN plasmids. The third is the resolvase, resP, which may resolve plasmid multimers that can lead to plasmid instability. pPSX is a small, stable cloning vector good for cloning and expression of a wide range of genes, including those from streptomycetes.

pPSY: a vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli.

pPSY is a 12kb cloning vector derived from the IncW plasmid R388, which provides a rapid and easy way to stably clone phenotypes encoded in DNA segments <10kb. In the present study three different genes were amplified by PCR, cloned into pGEM-T Easy and sub-cloned into the EcoRI site of pPSY. The first gene, vioA, is a FAD-dependent l-tryptophan amino acid oxygenase from the high G+C Gram-negative bacterium Chromobacterium violaceum. VioA is involved in the synthesis of the indolocarbazole antitumour antibiotic violacein. It was found that vioA was strongly expressed in Escherichia coli from its native promoter. Two other genes encoding recombinase A (recA) and an amylase (amyA), derived from the high G+C Gram-positive streptomycete, Streptomyces lividans, were also tested. Despite recA lacking its native promoter sequence, it was strongly expressed in E. coli using the lac promoter of pGEM-T Easy. Similar to vioA, S. lividansamyA was strongly expressed in E. coli from its native promoter. Unlike pGEM-T Easy, pPSY stably maintained all three genes without the requirement for antibiotic selection. These results demonstrate the applicability of pPSY as a stable amplicon cloning vector for the expression of heterologous genes in E. coli.

pPSX: a novel vector for the cloning and heterologous expression of antitumor antibiotic gene clusters.

A cosmid cloning vector has been constructed that demonstrates high levels of segregational stability in Escherichia coli K12. pPSX is a 14-kilobase vector derived from the IncW plasmid pR388. pPSX is highly stable in E. coli in the absence of antibiotic selection, even while expressing the toxic indolocarbazole antitumor antibiotic violacein. The incorporation of the lambdacos sequence enables construction of cosmid libraries with inserts ranging from 24 to 36kb. The inclusion of a lacZalpha multiple cloning site (MCS) allows blue/white screening. pPSX cosmids can be extracted from the host cell with commercial plasmid extraction kits facilitating downstream analysis, sequencing and sub-cloning. pPSX can be transferred to a variety of heterologous hosts by either electroporation or mobilization from E. coli S17-1. While it is unstable in non-E. coli hosts without antibiotic selection, heterologous host strains such as Rhodobacter sphaeroides and Pseudomonas stutzeri will maintain the plasmid under antibiotic selection to allow screening of expressed inserts. pPSX provides the benefits of large insert sizes with high stability to allow cloning of chemotherapeutic gene clusters in E. coli and a range of other heterologous hosts.

Isolation and characterization of integron-containing bacteria without antibiotic selection.

The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1 and intI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial colonies from the enrichment cultures were then isolated by using hydrophobic grid membrane filters and integrase-specific gene probes. Bacterial clones isolated by this technique were then confirmed to carry integrons by further testing by PCR and DNA sequencing. Integron-associated antibiotic resistance genes were detected in bacteria such as Escherichia coli, Aeromonas spp., Proteus spp., Morganella morganii, Shewanella spp., and urea-positive Providencia stuartii isolates from bovine fecal samples without the use of selective enrichment media containing antibiotics. Streptomycin and trimethoprim resistance were commonly associated with integrons. The advantages conferred by this methodology are that a wide variety of integron-containing bacteria may be simultaneously cultured in BPW enrichments and culture biases due to antibiotic selection can be avoided. Rapid and efficient identification, isolation, and characterization of antibiotic resistance-associated integrons are possible by this protocol. These methods will facilitate greater understanding of the factors that contribute to the presence and transfer of integron-associated antibiotic resistance genes in bacterial isolates from red meat production animals.

Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides.

A regulatory protein, PpaA, involved in photosystem formation in the anoxygenic phototrophic proteobacterium Rhodobacter sphaeroides has been identified and characterized in vivo. Based on the phenotypes of cells expressing the ppaA gene in extra copy and on the phenotype of the ppaA null mutant, it was concluded that PpaA activates photopigment production and puc operon expression under aerobic conditions. This is in contrast to the function of the PpaA homologue from Rhodobacter capsulatus, AerR, which acts as a repressor under aerobic conditions [Dong, C., Elsen, S., Swem, L. R. & Bauer, C. E. (2002). J Bacteriol 184, 2805-2814]. The expression of the ppaA gene increases several-fold in response to a decrease in oxygen tension, suggesting that the PpaA protein is active under conditions of low or no oxygen. However, no discernible phenotype of a ppaA null mutant was observed under anaerobic conditions tested thus far. The photosystem gene repressor PpsR mediates repression of ppaA gene expression under aerobic conditions. Sequence analysis of PpaA homologues from several anoxygenic phototrophic bacteria revealed a putative corrinoid-binding domain. It is suggested that PpaA binds a corrinoid cofactor and the availability or structure of this cofactor affects PpaA activity.

Construction of a physical and preliminary genetic map of Aeromonas hydrophila JMP636.

A physical and preliminary genetic map of the Aeromonas hydrophila JMP636 chromosome has been constructed. The topology of the genome was predicted to be circular as chromosomal DNA did not migrate from the origin during PFGE unless linearized by S1 nuclease. Cleavage of the chromosome with PacI and PmeI produced 23 and 14 fragments, respectively, and enabled calculation of the genome size at 4.5 Mb. Digestion of the chromosome with I-CeuI produced 10 fragments, indicating that 10 rrl (23S) genes were likely to be present. Hybridizations between DNA fragments generated with PacI, PmeI and I-CeuI were used to initially determine the relationship between these segments. To accurately map genes previously characterized from JMP636, the suicide vector pJP5603 was modified to introduce restriction sites for PacI and PmeI, producing pJP9540. Following cloning of genes into this vector and recombinational insertion into the JMP636 chromosome, PacI and PmeI cleavage determined the location of genes within macrorestriction fragments with the additional bands produced forming hybridization probes. From the data generated, it was possible to form a physical map comprising all the fragments produced by PacI and PmeI, and assign the contig of I-CeuI fragments on this map. The preliminary genetic map defines the location of six loci for degradative enzymes previously characterized from JMP636, while the locations of the 10 sets of ribosomal genes were assigned with less accuracy from hybridization data.

Manganous ions suppress photosynthesis gene expression in Rhodobacter sphaeroides.

The effect of manganous ions [Mn(II)] and ferrous ions [Fe(II)] on expression of photosynthesis genes in Rhodobacter sphaeroides was investigated. The presence of Mn(II) during phototrophic (anaerobic) and chemotrophic (aerobic) growth of R. sphaeroides caused a decrease in the amount of bacteriochlorophyll and carotenoid pigments which were synthesized and this was associated mainly with a decrease in the level of light-harvesting complex II. Mn(II) was shown to cause a decrease in expression of the puc operon, which encodes the polypeptides of light-harvesting complex II. Expression of the puc operon is controlled by the central repressor of photosynthesis gene expression, PpsR. In a ppsR mutant there was no effect of Mn(II) on photosynthesis gene expression. It is concluded that Mn(II) may act as a corepressor in the action of PpsR or act via an as yet uncharacterized protein that interacts with PpsR. In contrast to the effects of Mn(II), Fe(II) was required for high levels of photosynthesis gene expression. This requirement for Fe(II) was shown to be related to the regulation of hemA, a gene under the control of the transcriptional regulator, FnrL. Mn(II) did not affect FnrL-dependent gene expression.