No abstract

For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.

Triclosan is an antiseptic frequently added to items as diverse as soaps, lotions, toothpaste, and many commonly used household fabrics and plastics. Although wild-type Pseudomonas aeruginosa expresses the triclosan target enoyl-acyl carrier protein reductase, it is triclosan resistant due to expression of the MexABOprM efflux system. Exposure of a susceptible ⌬(mexAB-oprM) strain to triclosan selected multidrug-resistant bacteria at high frequencies. These bacteria hyperexpressed the MexCD-OprJ efflux system due to mutations in its regulatory gene, nfxB. The MICs of several drugs for these mutants were increased up to 500-fold, including the MIC of ciprofloxacin, which was increased 94-fold. Whereas the MexEF-OprN efflux system also participated in triclosan efflux, this antimicrobial was not a substrate for MexXY-OprM.Pseudomonas aeruginosa is a clinically significant pathogen, particularly in immunocompromised hosts (36). Infections caused by this bacterium are difficult to treat due to its many intrinsic and acquired antibiotic resistances. Intrinsic resistance is mostly attributable to the expression of several multidrug resistance (MDR) efflux systems. The P. aeruginosa genome (35) contains structural genes for at least 12 resistance nodulation type efflux systems, of which only 4, i.e., MexABOprM (27), MexCD-OprJ (26), MexEF-OprN (13), and MexXY (1, 21, 38), have been characterized. Exposure to selected substrates can select for their upregulated or constitutive expression (13,14,26,38).2-Hydroxyphenylethers are a class of compounds that exhibit broad-spectrum antimicrobial activity. Triclosan is the most potent and widely used member of this class (2, 5) and is used in hand soaps, lotions, toothpastes, and oral rinses, as well as in fabrics and plastics. It was long thought to act as a nonspecific "biocide" (29), but recent biochemical and genetic studies have shown that triclosan acts on a defined bacterial target in the fatty acid biosynthetic pathway, enoyl-acyl carrier protein (ACP) reductase (FabI) (7,9,10,12,18,20) or its homolog InhA in mycobacteria (18). Some bacteria possess triclosan-resistant enoyl-ACP reductase homologs (FabK), and to date P. aeruginosa is unique among gram-negative bacteria in that it possesses both triclosan-sensitive and -resistant enzymes (8). Alterations in FabI active-site residues confer resistance to triclosan (9,10,20). Of particular concern is that such amino acid changes selected by exposure to triclosan lead to cross-resistance with other antimicrobial agents (9), including clinically used front-line drugs, since some mutations leading to triclosan resistance in Mycobacterium smegmatis also caused resistance to isoniazid (18). Moreover, triclosan is a substrate of a multidrug efflux pump in clinical and laboratory Escherichia coli strains (19). We have recently shown that P. aeruginosa strain PAO1 is intrinsically resistant to triclosan by virtue of expression of the MexAB-OprM efflux pump (32), and the same is true for all strains of this species tested to date (...

No abstract

The structural genes for dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris Hildenborough were cloned as a 7.2-kbp SacII DNA fragment. Nucleotide sequencing indicated the presence of a third gene, encoding a protein of only 78 amino acids, immediately downstream from the genes for the ␣ and ␤ subunits (dsvA and dsvB). We designated this protein DsvD and the gene encoding it the dsvD gene. The ␣and ␤-subunit sequences are highly homologous to those of the dissimilatory sulfite reductase from Archaeoglobus fulgidus, a thermophilic archaeal sulfate reducer, which grows optimally at 83؇C. A gene with significant homology to dsvD was also found immediately downstream from the dsrAB genes of A. fulgidus. The remarkable conservation of gene arrangement and sequence across domain (bacterial versus archaeal) and physical (mesophilic versus thermophilic) boundaries indicates an essential role for DsvD in dissimilatory sulfite reduction and allowed the construction of conserved deoxyoligonucleotide primers for detection of the dissimilatory sulfite reductase genes in the environment.

Enterococcus faecalis was tested for the ability to persist in mouse peritoneal macrophages in two separate studies. In the first study, the intracellular survival of serum-passaged E. faecalis 418 and two isogenic mutants [cytolytic strain FA2-2(pAM714) and non-cytolytic strain FA2-2(pAM771)] was compared with that of Escherichia coli DH5α by infecting BALB/c mice intraperitoneally and then monitoring the survival of the bacteria within lavaged peritoneal macrophages over a 72-h period. All E. faecalis isolates were serum passaged to enhance the production of cytolysin. E. faecalis 418, FA2-2(pAM714), and FA2-2(pAM771) survived at a significantly higher level (P = 0.0001) than did E. coli DH5α at 24, 48, and 72 h. Internalized E. faecalis 418, FA2-2(pAM714), and FA2-2(pAM771) decreased 10-, 55-, and 31-fold, respectively, over the 72-h infection period, while internalizedE. coli DH5α decreased 20,542-fold. The difference in the rate of survival of E. faecalis strains and E. coli DH5α was most prominent between 6 and 48 h postinfection (P = 0.0001); however, no significant difference in killing was observed between 48 and 72 h postinfection. In the second study, additional E. faecalisstrains from clinical sources, including DS16C2, MGH-2, OG1X, and the cytolytic strain FA2-2(pAM714), were compared with the nonpathogenic gram-positive bacterium, Lactococcus lactis K1, for the ability to survive in mouse peritoneal macrophages. In these experiments, the E. faecalis strains and L. lactis K1 were grown in brain heart infusion (BHI) broth to ensure that there were equal quantities of injected bacteria. E. faecalis FA2-2(pAM714), DS16C2, MGH-2, and OG1X survived significantly better (P < 0.0001) than did L. lactis K1 at each time point. L. lactis K1 was rapidly destroyed by the macrophages, and by 24 h postinfection, viable L. lactis could not be recovered. E. faecalis FA2-2(pAM714), DS16C2, MGH-2, and OG1X declined at an equivalent rate over the 72-h infection period, and there was no significant difference in survival or rate of decline among the strains. E. faecalis FA2-2(pAM714), MGH-2, DS16C2, and OG1X exhibited an overall decrease of 25-, 55-, 186-, and 129-fold respectively, between 6 and 72 h postinfection. The overall reduction by 1.3 to 2.27 log units is slightly higher than that seen for serum-passaged E. faecalis strains and may be attributable to the higher level of uptake of serum-passaged E. faecalis than of E. faecalis grown in BHI broth. Electron microscopy of infected macrophages revealed that E. faecalis 418 was present within an intact phagocytic vacuole at 6 h postinfection but that by 24 h the infected macrophages were disorganized, the vacuolar membrane was degraded, and the bacterial cells had entered the cytoplasm. Macrophage destruction occurred by 48 h, and the bacteria were released. In conclusion, the results of these experiments indicate that E. faecaliscan persist for an extended period in mouse peritoneal macrophages.

A novel method for the identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a "standard") to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples.

51 that desulfoviridin, the dissimilatory sulfite reductase of sulfate-reducing bacteria of the genus Desulfovibrio, contains a third, y, subunit (1 1 kDa), in addition to the well-established a (50 kDa) and p (40 kDa) subunits, and an a 2 j z y z subunit structure has been proposed. Cloning and sequencing of the dsvC gene indicated it to encode a protein of 105 amino acids (1 1.9 kDa; y subunit). The finding that the dsvC gene, located on a 3.5-kb SacII fragment, is transcribed in both Escherichia coli and Desulfovihrio vulgaris as an mRNA of only 400-600 nucleotides, and that both the dsvA and dsvB genes are present on a 7. (66 kDa) subunits of the a&, assimilatory sulfite reductase from Salmonella typhimurium and Escherichia coli [l 1, 121, and the gene encoding the low-molecular-mass (24 kDa) assimilatory sulfite reductase of Desulfovibrio vulgaris Hildenborough [13]. Gene sequences and regulation of expression were also reported for asrA, asrB and asrC, encoding polypeptides of 4O,31 and 37 kDa, respectively, which form the sulfite reductase of S. typhiniurium [14], capable of reducing sulfite to sulfide under anaerobic conditions. The above indicate a lack of molecular biological information on the dissimilatory sulfite reductases of sulfate-reducing bacteria, in contrast to a wealth of data on the assimilatory enzymes. It appeared, therefore, worthwhle to clone and sequence the genes for desulfoviridin. This project began with the premise that the subunit structure was alp2, and with the expectation of finding an operon of about 2.5 kb, encoding both the a (50 kDa) and p(40 kDa) subunits. However, the cloning of a single gene for an 11-kDa protein reported in this paper presented a puzzle that was solved by the discovery of the y subunit by Pierik et al. [2]. MATERIALS AND METHODS MaterialsAll enzymes used for cloning and dideoxy sequencing were obtained from Pharmacia. Immunoblot staining reagents, nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, and anti-[mouse IgG(H + L)] serum linked to alkaline phosphatase were from Promega. Antibodies against the SDSdenatured c1, p and y desulfoviridin subunits, induced in mice (j? and y) and rabbits (M), were gifts of Drs A. J. Pierik and W.