Vectors to express foreign genes and techniques to monitor gene expression in Pseudomonads.

Improved tools for Pseudomonas research include small, broad-host-range vectors that allow regulated expression from the lac operon and T7 promoters whose biology is well understood and adaptable to many bacteria. To facilitate studies on gene regulation, tracking and monitoring of bacteria in diverse environments, and the construction of biosensors, various reporter genes with versatile assay formats have been developed that can be delivered on plasmid, transposon and integration-proficient vectors.

Correlation of imaging techniques to histopathology in patients with diabetic foot syndrome and clinical suspicion of chronic osteomyelitis. The role of high-resolution ultrasound.

OBJECTIVE: To investigate the role of ultrasound in the diagnosis of osteomyelitis in the diabetic foot compared with magnetic resonance imaging (MRI), bone scintigraphy (BS), and plain film radiography (PFR). RESEARCH DESIGN AND METHODS: We investigated 19 consecutive diabetic patients (2 women, 17 men, age 60.7 +/- 9.8 years, BMI 27.0 +/- 3.8 kg/m2) with clinical suspicion of bone infection of the foot. A high-resolution ultrasound system (Esaote/Biosound, Munich) with a linear array transducer up to 13.0 MHz was used. The prospective and blinded results of each method were compared with histopathology as the reference method after metatarsal resection. RESULTS: In 14 of 19 patients, histopathology confirmed osteomyelitis. Ultrasound showed a sensitivity of 79% (PFR, 69%; BS, 83%; MRI, 100%), a specificity of 80% (PFR, 80%; BS, 75%; MRI, 75%), a positive predictive value of 92% (PFR, 90%; BS, 91%; MRI, 93%), and a negative predictive value of 57% (PFR, 50%; BS, 60%; MRI, 100%). CONCLUSIONS: Our data indicate that ultrasound might have a better diagnostic power for detecting chronic osteomyelitis in the diabetic foot than PFR and has similar sensitivity and specificity as BS. MRI is superior to the other three methods. We conclude that the use of ultrasound in the management of the diabetic foot is worthy of further investigation.

A Burkholderia pseudomallei colony variant necessary for gastric colonization.

UNLABELLED: Diverse colony morphologies are a hallmark of Burkholderia pseudomallei recovered from infected patients. We observed that stresses that inhibit aerobic respiration shifted populations of B. pseudomallei from the canonical white colony morphotype toward two distinct, reversible, yet relatively stable yellow colony variants (YA and YB). As accumulating evidence supports the importance of B. pseudomallei enteric infection and gastric colonization, we tested the response of yellow variants to hypoxia, acidity, and stomach colonization. Yellow variants exhibited a competitive advantage under hypoxic and acidic conditions and alkalized culture media. The YB variant, although highly attenuated in acute virulence, was the only form capable of colonization and persistence in the murine stomach. The accumulation of extracellular DNA (eDNA) was a characteristic of YB as observed by 4',6-diamidino-2-phenylindole (DAPI) staining of gastric tissues, as well as in an in vitro stomach model where large amounts of eDNA were produced without cell lysis. Transposon mutagenesis identified a transcriptional regulator (BPSL1887, designated YelR) that when overexpressed produced the yellow phenotype. Deletion of yelR blocked a shift from white to the yellow forms. These data demonstrate that YB is a unique B. pseudomallei pathovariant controlled by YelR that is specifically adapted to the harsh gastric environment and necessary for persistent stomach colonization. IMPORTANCE: Seemingly uniform populations of bacteria often contain subpopulations that are genetically identical but display unique characteristics which offer advantages when the population is faced with infrequent but predictable stresses. The pathogen Burkholderia pseudomallei is capable of forming several reversible colony types, and it interconverted between one white type and two yellow types under certain environmental stresses. The two yellow forms exhibited distinct advantages in low-oxygen and acidic environments. One yellow colony variant was the only form capable of chronic stomach colonization. Areas of gastric infection were marked by bacteria encased in a DNA matrix, and the yellow forms were able to produce large amounts of extracellular DNA in vitro. We also identified the regulator in control of yellow colony variant formation. These findings demonstrate a role in infection for colony variation and provide a mechanism for chronic stomach colonization-a frequently overlooked niche in melioidosis.

Improved broad-host-range lac-based plasmid vectors for the isolation and characterization of protein fusions in Pseudomonas aeruginosa.

Several new broad-host-range vectors for the construction of protein fusions to the Escherichia coli lacZ gene have been developed. In all of the constructs, a multiple cloning site (MCS) containing unique restriction sites is located upstream of lac operon segments whose lacZ genes lack translational start signals. Some of the vectors (pPZ10, pPZ20 and pPZ30) also contain transcriptional terminators upstream of the MCS. The new vectors allow the fusion of genes to lacZ in all translational reading frames. Due to a higher copy number they allow direct screening in E. coli for weakly expressed foreign promoters. Their usefulness for gene analysis in Pseudomonas aeruginosa was demonstrated by construction and expression of a regA'::'lacZ-encoded protein fusion.

Escherichia-Pseudomonas shuttle vectors derived from pUC18/19.

Two new broad-host-range plasmid vectors, pUCP18 and pUCP19, which are stably maintained in Escherichia coli and Pseudomonas aeruginosa have been constructed. The plasmids are based on the E. coli pUC18 and pUC19 vectors and possess all their features: (i) convenient direct screening of recombinants; (ii) versatile multiple cloning site; (iii) use as sequencing and expression vectors; (iv) small size; and (v) intermediate to high copy number.

Two plasmids, X1918 and Z1918, for easy recovery of the xylE and lacZ reporter genes.

Two plasmids, X1918 and Z1918, were constructed which contain the promoter-less xylE and lacZ reporter genes flanked symmetrically by the multiple cloning site (MCS) from pUC19. These cassettes can easily be derived from the multicopy plasmid, pUC1918.

An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa.

A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZ alpha allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered to contain few restriction sites to gain greater access to restriction sites within cloned DNA fragments, thus facilitating their genetic manipulation. The usefulness of the system was demonstrated by chromosomal integration of a newly constructed xylE::GmR fusion cassette into the glpD gene of Pseudomonas aeruginosa.

Utilization of a mini-Dlac transposable element to create an alpha-complementation and regulated expression system for cloning in Pseudomonas aeruginosa.

A lac-based alpha-complementation and expression system was developed for use in molecular cloning in Pseudomonas aeruginosa. A bacteriophage D3112-based mini-Dlac transposable element, containing the lacIq-regulated lacZ delta M15 gene next to a selectable marker, was constructed. Mixed D3112 lysates were used to transduce P. aeruginosa PAO1, and derivatives containing randomly inserted chromosomal copies of the mini-Dlac element were obtained. Transformation of the PAO1::mini-Dlac transductants with the broad-host-range vector, pUCP19, led to the formation of blue colonies on indicator medium in the presence of inducer. In contrast, transformants harboring the pUCP19 derivative pCDO, containing the catechol-2,3-dioxygenase (C23O)-encoding xylE gene under lac promoter control, were white on the same medium. Expression of xylE was tightly controlled by single-copy mini-Dlac-encoded lac repressor and in induced cultures was increased more than 100-fold over that observed in uninduced cultures. The usefulness of the system for molecular cloning in P. aeruginosa was demonstrated by ligating size-fractionated PAO1 chromosomal fragments into pUCP19, followed by transformation of the newly isolated PAO1::mini-Dlac host. All randomly chosen white colonies contained recombinant plasmids, with inserts of the correct size range, while blue colonies contained pUCP19 alone. The functionality of the system was also shown in another frequently studied strain, PA103.

Cloning and sequence analysis of the fur gene encoding an iron-regulatory protein of Neisseria meningitidis.

The fur gene, encoding an iron-regulatory protein in Neisseria meningitidis strain B16B6, has been cloned and sequenced. Its product showed a high degree of homology to known Fur sequences.

Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa.

The nucleotide sequence of the 1.9-kb PstI fragment from pRO1614, that allows stable maintenance of pMB1 (ColE1)-based cloning vectors in Pseudomonas, was determined. This fragment encodes a putative origin of replication (ori), a replication-controlling protein, and the C terminus of the Tn3 beta-lactamase-encoding gene. Improved versions of the broad-host-range plasmid vectors, pUCP18 and pUCP19, were constructed by deletion of nonessential DNA or replacement of nonessential DNA with an antibiotic-resistance cassette.

A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants.

An improved method for gene replacement in Pseudomonas aeruginosa was developed. The method employs several new gene replacement vectors that incorporate (1) the counterselectable sacB marker, (2) a lacZ alpha-allele for blue-white screening, (3) the pUC18/19 vectors multiple cloning site with 10 unique restriction sites, (4) an oriT for conjugation-mediated plasmid transfer and (5) carbenicillin, gentamicin (Gm) and tetracycline selectable markers. A cassette was constructed that contains a GmR selectable marker next to the green fluorescent protein structural gene, with both markers being flanked by Flp recombinase target (FRT) sites. The FRT cassette was used to insertionally inactivate the cloned P. aeruginosa pabC gene encoding aminodeoxychorismate lyase. After conjugal transfer into P. aeruginosa, plasmid integrants were selected, and deletion of unwanted DNA sequences was promoted by sucrose counterselection. The FRT cassette was excised with high frequencies (close to 100%) from the chromosome after conjugal transfer of a Flp recombinase-expressing plasmid; this sacB-containing plasmid was subsequently cured by sucrose counterselection, resulting in an unmarked P. aeruginosa delta pabC strain.

Cloning and characterization of the Neisseria meningitidis asd gene.

The asd mutants of Gram- and some Gram+ bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, i.e., mammalian tissues, they will undergo lysis. This has previously been exploited to develop vaccine strains of Salmonella typhimurium and Streptococcus mutans. As a first step for the development of a biosafe Neisseria meningitidis laboratory strain, we have cloned the asd from wild-type strain B16B6 by complementation of an Escherichia coli asd mutant. By subcloning and insertion mutagenesis, the N. meningitidis asd was localized to a 1.5-kb DNA fragment. In a T7 RNA polymerase-T7 promoter expression system, a 38-kDa protein was strongly expressed from this DNA fragment. The N-terminal amino acid (aa) sequence was deduced from the nucleotide sequence, which was determined with the help of an in-frame Asd'::'LacZ protein fusion. A comparison of the N-terminal aa of the Asd proteins from N.meningitidis and E. coli revealed 70% identity, suggesting that the Asd protein may be highly conserved among Gram- bacteria.

Construction and use of low-copy number T7 expression vectors for purification of problem proteins: purification of mycobacterium tuberculosis RmlD and pseudomonas aeruginosa LasI and RhlI proteins, and functional analysis of purified RhlI.

Purification of proteins from Escherichia coli under native conditions is often hampered by inclusion-body formation after overexpression from T7 promoter-based expression vectors. This is probably due to the relatively high copy number of the ColE1-based expression vectors. To circumvent these problems, the low-copy-number pViet and pNam expression vectors were constructed. These vectors contain the pSC101 origin of replication and allow the expression of oligohistidine and intein chitin-binding domain fusion proteins, respectively. Since pViet and pNam do not replicate in E. coli B strains, an E. coli K-12 host strain [SA1503(DE3)] was constructed. This strain is defective in the Lon and OmpT proteases and allows IPTG-inducible expression of recombinant proteins from the T7 promoter. The new vectors were successfully tested by purification of three very insoluble proteins (RmlD, LasI and RhlI) under non-denaturing conditions, and all three proteins retained enzymatic activity. The purified hexahistidine (His6)-tagged Pseudomonas aeruginosa RhlI protein was subjected to more detailed analyses, which indicated that (1) only butyryl-acyl carrier protein (ACP) and S-adenosylmethionine (SAM) were required for synthesis of N-butyryl-L-homoserine lactone; (2) when present at physiological concentrations, butyryl-coenzyme A and NADPH were not substrates for RhlI; (3) RhlI was able to synthesize N-hexanoyl-L-homoserine lactone from hexanoyl-ACP and SAM; (4) RhlI was able to direct synthesis of N-butyryl-L-homoserine lactone from crotonyl-ACP in a reaction coupled to purified P. aeruginosa FabI (enoyl-ACP reductase).

ASD-GFP vectors for in vivo expression technology in Pseudomonas aeruginosa and other gram-negative bacteria.

We describe the construction of promoter probe vectors designed for identification of bacterial genes induced in vitro and/or in vivo and for measurement of gene expression levels for in vivo expression technology. These plasmids use the Pseudomonas aeruginosa aspartate beta-semialdehyde dehydrogenase (asd) gene as a selectable marker and beta-galactosidase (pIVPRO, 10.88 kb) or mutant green fluorescent protein with enhanced fluorescence properties (mut3GFP, pIVET-GFP, 5.48 kb) as reporter gene systems. The proposed strategies can be adapted for use in most Gram-negative bacteria.

Triclosan: a widely used biocide and its link to antibiotics.

Triclosan is the active ingredient in a multitude of health care and consumer products with germicidal properties, which have flooded the market in recent years in response to the public's fear of communicable bacteria. Although originally thought to kill bacteria by attacking multiple cellular targets, triclosan was recently shown to target a specific bacterial fatty acid biosynthetic enzyme, enoyl-[acyl-carrier protein] reductase, in Gram-negative and Gram-positive bacteria, as well as in the Mycobacteria. Triclosan resistance mechanisms include target mutations, increased target expression, active efflux from the cell, and enzymatic inactivation/degradation. These are the same types of mechanisms involved in antibiotic resistance and some of them account for the observed cross-resistance with antibiotics in laboratory isolates. Therefore, there is a link between triclosan and antibiotics, and the widespread use of triclosan-containing antiseptics and disinfectants may indeed aid in development of microbial resistance, in particular cross-resistance to antibiotics.

Contribution of multidrug efflux pumps to multiple antibiotic resistance in veterinary clinical isolates of Pseudomonas aeruginosa.

The contribution of efflux pumps to multidrug resistance in 12 Pseudomonas aeruginosa isolates from various animal sources was assessed. Western immunoblot analyses demonstrated that all twelve isolates expressed significant levels of the MexAB OprM efflux system whereas two isolates simultaneously expressed the MexEF OprN or MexXY systems, respectively. One strain contained a single mutation in mexR, a regulator of mexAB-oprM expression, that did not adversely affect the MexR amino acid sequence, and three isolates contained the same, single base change in the mexA-mexR intergenic region. The MexXY-expressing strain contained two base substitutions in its mexZ regulatory gene which did not alter the MexR sequence.