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Research has been conducted on the TetA efflux pump protein. The study of the site where tetracyclin binds to this pump protein has been carried out and the results indicate that when chelated to tetracyclin Fe (super)2+ cleaves TetA backbone at glutamine 225 in transmembrane helix 7.

The Pseudomonas fluorescens transcription activator AdnA has been found to be necessary for motility and adhesion. Both are in a gene showing homology to the NtrC/NifA family of transcription activators. The phenotypes seen are caused by lack of AdnA and are not the result of other mutation or polar effects of mutations in adnA on other genes. The insertions Pf0-5 and Pf0-10 have been localized. The disrupted genes are now known to be the same ORF. Recently found data indicate that in P. aeruginosa, the FliD protein, at the tips of flagella, mediates a specific interaction with mucins.

The transcription factor AdnA of Pseudomonas fluorescens allows the organism to spread and survive in soil. This is important because this organism is considered to be beneficial in treating plant diseases, decontaminating soil, and improving plant nutrition.

Research has been conducted on the MarA proteins in Escherichia coli. Results indicate that the mar locus mediates a global stress response that involves chromosomal genes.

The multiple antibiotic resistance locus (mar) of Escherichia coli regulates intrinsic susceptibility to multiple antibiotics, organic solvents, oxidative stress agents and household disinfectants. MarR negatively regulates expression of the multiple antibiotic resistance operon (marRAB) in Escherichia coli. Research was conducted which revealed that sodium salicylate, plumbagin, 2,4-dinitrophenol and menadione-inducers of the marRAB operon in whole cells, interfered with the repressor activity of Mar R in vitro.

The existence of mar-like regulatory drug resistance response was investigated in Mycobacterium smegmatis by analyzing antimicrobial vulnerability in cells that express the cloned Escherichia coli marA gene. The expression resulted in increased resistance to multiple antimicrobial agents, such as chloramphenicol, tetracycline, ethambutol, isoniazid and rifampicin. Changes in resistance did not materialize when the plasmid bearing marA was lost.

A study using site-directed mutagenesis was conducted to examine the role of glutamate residues located within putative transmembrane helices in TetA(P)-mediated tetracycline efflux. The results indicate that the negatively charged residues that seem to be located within or near the membrane play an essential role in the function of TetA(P). Moreover, different helices in different Tet proteins may have homologous functions.

A research study was conducted regarding the role of acr and multiple antibiotic resistance (mar) loci in organic solvent tolerance mediated by expression of marA, soxS or robA in Escherichia coli (E coli). The methods used were the DNA manipulations and organic solvent tolerance analysis. Results showed that marA, soxS and robA can independently create organic solvent tolerance in E coli with the presence of acrAB locus.

Transcriptional fusion of marOII-lacZ into the chromosome of wild-type and purified marR mutants was examined to determine the factors affecting the expression of the marRAB operon in Escherichia coli. Wild-type cells exhibited a two- to seven-fold increase in LacZ expression, while mar-deleted cells exhibited a 20-fold increase. Results of the fusion of MarR with a fusion protein MarE indicated repressor ability and a dissociation constant of 5 x 10(sub -9) M, suggesting that repression in cells are reversed to facilitate the response of the bacteria to stress.

A study of the participation of non-marRAB chromosomal loci in chromosomal multiple antibiotic resistance (Mar) phenotype using TrophoA and TnlacZ elements helps study mar-sensitive fusions in a specially designed Escherichia coli recipient that has a temperature-sensitive plasmid carrying the mar locus and the mar RAB operon. Five mar-regulated gene fusions are detected, with beta-galactosidase expression being stimulated in two of them and alkaline phosphate expression being suppressed in the other three. Occurrence of these changes due to marA alone indicates that MarA is the regulatory protein that leads to enhanced resistance to multiple antibiotics.

Two adhesion-defective mutants, Pf0-10 and Pf0-15, with different Tn5 insertion positions, are isolated from Pseudomonas fluoresences. Pf0-15 contains high concentrations of a 34-kDa outer membrane protein and flagella for mobility, unlike Pf0-10 which lacks both features. The mutants exhibit discrepancies in initial attachment steps but reveal a slow final binding phase.

Three pleiotropic mutations of the 34 min mar locus of Escherichia coli activate the marRAB operon that probably triggers antibiotic resistance and oxidative stress genes by relieving the repressive effects of MarR protein. the pleiotropic effects are reversed to varying degrees by marR+ gene or a multicopy plasmid. The underlying mechanisms of these marR1, soxQ1 and cfxB1 mutants are possibly similar as shown by studies of RNA expression.

An analysis of the influence of salicylate on the expressin of the marRAB operon reveals that salicylate activates expression of the marRAB operon, which indicates involvement of the mar locus in phenotypic antibiotic resistance (PAR). Development in salicylate in both mar-lacking and mar-containing strains leads to enhanced antibiotic OmpF, enhanced micF transcription and reduced permeation of the external membrane to cephaloridine. However, these changes occur more in cells which contain mar.

Escherichia coli multiple antibiotic resistance locus from a 7,800 base-pair region in the chromosomal DNA has been sequenced. A sequence within this region has the potential to be a divergent promoter which includes a segment with two pairs ofdirect repeats, marO. Flanking marO are putative open reading frames (ORF) three on either side. Mutations in the downstream ORFs were characterized.

A Tn5 insertion which led to increased susceptibility to multiple drugs, including tetracycline, chloramphenicol, nalidixic acid, erythromycin, spectinomycin, norfloxacin and novobiocin, was identified in Escherichia coli. Cloning and sequence analysis showed that this locus is identical to prc gene, which map at min 40.4. The prc product has beenlinked to proteolytic processing of penicillin-binding protein 3. Moreover, theresults indicate that the prc gene product affects sensitivity to multiple antibiotics possibly by influencing cell permeability.

The role of the aspartate 15 residue of the class B tetracycline efflux protein Tet was studied, using site-directed mutagenesis in Escherichia coli. Replacement of aspartate by tyrosine, histidine or asparagine resulted in a reduction in tetracycline resistance. The tyrosine and histidine substitutions lowered the Vmax of tetracycline transport but did not change the Km. On the other hand, the asparagine substitution increased the Km but did not change the Vmax. Therefore, Asp-15 could play a role in Tet efflux.

Results show that tetracycline resistance, mediated by the transmembrane helix 8 of the tetracyline efflux protein Tet(B), is abolished when Gly247 is substituted with aspartate or aspergine. However, partial restoration is accomplished by second site suppressor mutations in transmembrane helix 5 or in cytoplasmic loops.