John M. Pemberton

An improved suicide vector for construction of chromosomal insertion mutations in bacteria

We have constructed an R6K-based suicide vector (pJP5603) that requires a trans supply of the pir-encoded pi protein of plasmid R6K for replication. Therefore, efficient plasmid suicide results upon transfer to bacteria not harbouring pir. The 3.1-kb vector encodes kanamycin resistance and is mobilizable. When used in conjunction with a JM109 strain carrying pir, it has nine unique restriction sites available for alpha-complementation cloning. Vector functionality was demonstrated in Rhodobacter sphaeroides.

Isolation and Characterization of Integron-Containing Bacteria without Antibiotic Selection

ABSTRACT 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.

Domain Structure, Oligomeric State, and Mutational Analysis of PpsR, the Rhodobacter sphaeroides Repressor of Photosystem Gene Expression

The transcription factor PpsR from the facultative photoheterotroph Rhodobacter sphaeroides is involved in repression of photosystem gene expression under aerobic growth conditions. We have isolated a number of spontaneous mutations as well as constructed directed mutations and deletions in ppsR. Repressor activities and the oligomeric state of the wild-type and mutant proteins were assayed. Our results suggest that the wild-type PpsR exists in cell extracts as a tetramer. Analysis of the PpsR mutants confirmed that the carboxy-terminal region of PpsR (residues 400 to 464) is involved in DNA binding. The central region of the protein (residues 150 to 400) was found to contain two PAS domains (residues 161 to 259 and 279 to 367). PAS domains are ubiquitous protein modules involved in sensory transduction as well as in protein-protein interactions. All spontaneously isolated mutations, which significantly impaired repressor activity and which mapped outside the DNA binding region, were positioned in the PAS domains. None of these, however, affected the overall oligomeric state. This implies that the conformation of the PAS domains within the tetramer is critical for repressor activity. Upstream of the first PAS domain resides a putative glutamine-rich hinge (residues 127 to 136) that connects the first PAS domain to the amino-terminal region (residues 1 to 135). The role of the amino terminus of PpsR is not obvious; however, extended deletions within this region abolish repressor activity, thus suggesting that the amino terminus is essential for structural integrity of the protein. We present a model of the domain architecture of the PpsR protein according to which PpsR is comprised of three regions: the carboxy terminus responsible for DNA binding, the central region primarily involved in protein oligomerization and possibly signal sensing, and the amino terminus of unknown function. This model may prove useful for determining the mode of PpsR action.

A gene from the photosynthetic gene cluster ofRhodobacter sphaeroides inducestrans suppression of bacteriochlorophyll and carotenoid levels inR. sphaeroides andR. capsulatus

ARhodobacter sphaeroides gene (pps) inducestrans suppression of bacteriochlorophyll (Bch) and carotenoid (Crt) levels in bothR. sphaeroides andR. capsulatus. It also induces suppression of Crt levels in aParacoccus denitrificans strain carrying the Crt genes ofR. sphaeroides. The gene is located approximately 11 kilobases fromcrtA in the photosynthetic gene cluster. Crt suppression bypps is quantitatively different from that caused by an absence of mature Bch.

Cloning and heterologous expression of the violacein biosynthesis gene cluster fromChromobacterium violaceum

The entire biosynthetic pathway for the synthesis of the antibiotic pigment violacein is encoded on a 14.5 kilobase (kb) fragment of theChromobacterium violaceum genome. When cloned intoEscherichia coli, pigment synthesis is strongly expressed, as it is in a wide range of Gram-negative bacteria. Transposon mutagenesis resulted in a number of different phenotypes that correlated with transposon insertion sites in the gene cluster.

Regulation of photosynthetic gene expression in purple bacteria

Purple phototrophic bacteria have the ability to capture and use sunlight efficiently as an energy source. In these organisms, photosynthesis is carried out under anaerobic conditions. The introduction of oxygen into a culture growing phototrophically results in a rapid decrease in the synthesis of components of the photosynthetic apparatus and a change to an alternative source of energy, usually derived from the degradation of organic compounds under aerobic conditions (chemoheterotrophy). Switching back and forth between anaerobic (photosynthetic) and aerobic growth requires tight regulation of photosynthetic gene expression at the molecular level. Initial experiments by Cohen-Bazire et al. (1957) showed quite clearly that the regulation of photosynthetic gene expression was in response to two environmental stimuli. The most potent stimulus was oxygen; its presence shut down production of photosynthetic pigments very rapidly. To a lesser extent photosynthetic gene expression responded to light intensity. Low light intensity produced high levels of photosynthetic pigments; high light intensities caused a decrease, but the effect was less dramatic than that observed for oxygen. Since these initial observations were made in Rhodobacter sphaeroides some forty years ago, a great deal has been revealed as to the nature of the genes that encode the various components of the photosynthetic apparatus. Recent progress in the understanding of the regulation of expression of these genes in R. sphaeroides and Rhodobacter capsulatus is the subject of this review.

Cloning and characterization of an albicidin resistance gene from Klebsiella oxytoca

A DNA fragment containing a gene for resistance to the antibiotic albicidin was isolated from Klebsiella oxytoca and shown to be expressed in Escherichia coli, where it also protected bacteriophage T7 replication from inhibition by albicidin. In vivo translation analysis demonstrated that the cloned 2.2kb DNA fragment coded for a 36 kiloDalton (kD) protein and a 25 kD protein.

Cloning and expression of anAeromonas hydrophila chitinase gene inescherichia coli

The gene encoding an extracellular chitinase fromAeromonas hydrophila has been cloned and expressed inEscherichia coli. Plasmid pJP2512 contained the smallest DNA insert (3.9 kb) producing chitinase. The chitinase gene is transcribed from its own promoter, producing a protein of Mr 96,000. The chitinase open reading frame, an estimated 2.6 kb in length, has been subcloned to a 3.0 kb fragment; however, this fragment does not carry the functional chitinase promoter. InE. coli the chitinase enzyme is unable to transverse the outer membrane, being secreted across the cytoplasmic membrane and accumulating in the periplasmic space.

Stable albicidin resistance in Escherichia coli involves an altered outer-membrane nucleoside uptake system

Albicidin blocked DNA synthesis in intact cells of a PolA- EndA- Escherichia coli strain, and in permeabilized cells supplied with all necessary precursor nucleotides, indicating a direct effect on prokaryote DNA replication. Replication of phages T4 and T7 was also blocked by albicidin in albicidin-sensitive (Albs) but not in albicidin-resistant (Albr) E. coli host-cells. All stable spontaneous Albr mutants of E. coli simultaneously became resistant to phage T6. The locus determining albicidin sensitivity mapped at tsx, the structural gene for an outer-membrane protein used as a receptor by phage T6 and involved in transport through the outer membrane of nucleosides present at submicromolar extracellular concentrations. Albicidin does not closely resemble a nucleoside in structure. However, Albs E. coli strains rapidly accumulated both nucleosides and albicidin from the surrounding medium whereas the Albr mutants were defective in uptake of nucleosides and albicidin at low extracellular concentrations. An insertion mutation blocking Tsx protein production also blocked albicidin uptake and conveyed albicidin resistance. Albicidin supplied at approximately 0.1 microM blocked DNA replication within seconds in intact Albs E. coli cells, but a 100-fold higher albicidin concentration was necessary for a rapid inhibition of DNA replication in permeabilized cells. We conclude that albicidin is effective at very low concentrations against E. coli because it is rapidly concentrated within cells by illicit transport through the tsx-encoded outer-membrane channel normally involved in nucleoside uptake. Albicidin resistance results from loss of the mechanism of albicidin transport through the outer membrane.

Cloning of carotenoid biosynthesis genes from Rhodopseudomonas sphaeroides

Two cosmids, pJP1433 and pJP1488, have been constructed which carry between them 60 kilobases of the central section of the photosynthesis region ofRhodopseudomonas sphaeroides. Within this region is a segment of some 15 kilobases which encompasses the known carotenoid biosynthesis genesCrtA,CrtB,CrtC,CrtD,CrtE, andCrtF.