Annihilation of exceptional points from different Dirac valleys in a 2D photonic system.

Topological physics relies on Hamiltonian's eigenstate singularities carrying topological charges, such as Dirac points, and - in non-Hermitian systems - exceptional points (EPs), lines or surfaces. So far, the reported non-Hermitian topological transitions were related to the creation of a pair of EPs connected by a Fermi arc out of a single Dirac point by increasing non-Hermiticity. Such EPs can annihilate by reducing non-Hermiticity. Here, we demonstrate experimentally that an increase of non-Hermiticity can lead to the annihilation of EPs issued from different Dirac points (valleys). The studied platform is a liquid crystal microcavity with voltage-controlled birefringence and TE-TM photonic spin-orbit-coupling. Non-Hermiticity is provided by polarization-dependent losses. By increasing the non-Hermiticity degree, we control the position of the EPs. After the intervalley annihilation, the system becomes free of any band singularity. Our results open the field of non-Hermitian valley-physics and illustrate connections between Hermitian topology and non-Hermitian phase transitions.

Maize chromomethylase Zea methyltransferase2 is required for CpNpG methylation.

A cytosine DNA methyltransferase containing a chromodomain, Zea methyltransferase2 (Zmet2), was cloned from maize. The sequence of ZMET2 is similar to that of the Arabidopsis chromomethylases CMT1 and CMT3, with C-terminal motifs characteristic of eukaryotic and prokaryotic DNA methyltransferases. We used a reverse genetics approach to determine the function of the Zmet2 gene. Plants homozygous for a Mutator transposable element insertion into motif IX had a 13% reduction in methylated cytosines. DNA gel blot analysis of these plants with methylation-sensitive restriction enzymes and bisulfite sequencing of a 180-bp knob sequence showed reduced methylation only at CpNpG sites. No reductions in methylation were observed at CpG or asymmetric sites in heterozygous or homozygous mutant plants. Our research shows that chromomethylase Zmet2 is required for in vivo methylation of CpNpG sequences.

ZMPP2, a novel type-2C protein phosphatase from maize.

A cDNA clone was selected as a candidate for the catalytic subunit of phospho-pyruvate dehydrogenase phosphatase (PDP) by screening a Zea mays expressed sequence tag database with the bovine PDP deduced amino acid sequence. Both strands of the cDNA were completely sequenced. The maize clone contains an open reading frame of 1098 base pairs that encodes a polypeptide of 40 127 Da, ZMPP2. The deduced amino acid sequence of ZMPP2 contains the five PP2C signature domains, as does PDP. However, the expression pattern of ZMPP2, determined by reverse transcriptase-polymerase chain reaction, was different from those of the maize pyruvate dehydrogenase E1 alpha subunit and pyruvate dehydrogenase kinase. Additionally, the predicted subcellular location of ZMPP2 is cytoplasmic, while the pyruvate dehydrogenase complex, regulated by reversible phosphorylation, is mitochondrial. Thus, ZMPP2 is a PP2C-type protein phosphatase related to but distinct from PDP.

Characterization of a gene from Zea mays related to the Arabidopsis flowering-time gene LUMINIDEPENDENS.

The molecular biology of flowering has been most extensively studied in the quantitative long-day plant Arabidopsis thaliana. The Arabidopsis LUMINIDEPENDENS (LD) gene encodes a potential transcriptional regulator that acts as a positive effector of flowering, at least in part through suppression of the floral inhibitor gene FLC. As an initial step to explore the conservation of the molecular mechanisms of flowering among plants of various flowering habits, and to further investigate the molecular action(s) of LD, we have identified a gene from maize (Zea mays) that is closely related to Arabidopsis LD. The major product of this gene, which we have designated ZmLD for Zea mays LUMINIDEPENDENS, contains four conserved regions that may constitute functionally important components of the LD proteins. One of these regions closely resembles the canonical homeodomain. The ZmLD gene exists as a single copy in the maize genome, and generates a major ca. 4.0 kb transcript, and a minor ca. 2.6 kb transcript that results from alternative transcriptional termination. The 4.0 kb ZmLD alpha transcript accumulated to highest levels in proliferative tissues, including the shoot apex and developing inflorescences. Expression of ZnLD alpha under control of the Arabidopsis LD promoter in transgenic Arabidopsis caused developmental defects similar to those conferred by loss-of-function mutations in a class of genes involved in maintaining the proliferative nature of the shoot, inflorescence, and floral meristems. These effects were not influenced markedly by the activities of the Arabidopsis LD or FLC genes. We consider the implications for the conservation of LD function between maize and Arabidopsis.

Developmental patterns of chromatin structure and DNA methylation responsible for epigenetic expression of a maize regulatory gene.

Epigenetic regulatory mechanisms heritably alter patterns of gene expression without changes in DNA sequence. Epigenetic states are often correlated with developmentally imposed alterations in genomic DNA methylation and local chromatin structure. Pl-Blotched is a stable epigenetic allele of the maize anthocyanin regulatory gene, purple plant1(pl). Pl-Blotched plants display a variegated pattern of pigmentation that contrasts sharply with the uniformly dark purple pigmentation of plants carrying the dominant Pl-Rhoades allele. Previously, we showed that the lower level of pigmentation in Pl-Blotched is correlated with lower pl mRNA levels and increased DNA methylation at some sites. To explore how DNA methylation, chromatin structure, and developmental stage might contribute to the expression of Pl-Blotched, we used methylation-sensitive restriction enzymes and DNaseI sensitivity assays to compare the methylation status and chromatin structure of Pl-Blotched and Pl-Rhoades at different stages in development. Both alleles exhibit developmentally sensitive changes in methylation. In Pl-Blotched, methylation of two diagnostic HpaII/MspI sites increases progressively, coincident with the juvenile-to-adult transition in growth. In seedlings, the chromatin encompassing the coding region of the gene is less sensitive to DNaseI digestion in Pl-Blotched than in Pl-Rhoades. Developmental maturation from seedling to adult is accompanied by expansion of this closed chromatin domain to include the promoter and downstream flanking sequences. We provide evidence to show that chromatin structure, rather than DNA methylation, is the primary epigenetic determinant for the phenotypic differences between Pl-Blotched and Pl-Rhoades.

Conserved plant genes with similarity to mammalian de novo DNA methyltransferases.

DNA methylation plays a critical role in controlling states of gene activity in most eukaryotic organisms, and it is essential for proper growth and development. Patterns of methylation are established by de novo methyltransferases and maintained by maintenance methyltransferase activities. The Dnmt3 family of de novo DNA methyltransferases has recently been characterized in animals. Here we describe DNA methyltransferase genes from both Arabidopsis and maize that show a high level of sequence similarity to Dnmt3, suggesting that they encode plant de novo methyltransferases. Relative to all known eukaryotic methyltransferases, these plant proteins contain a novel arrangement of the motifs required for DNA methyltransferase catalytic activity. The N termini of these methyltransferases contain a series of ubiquitin-associated (UBA) domains. UBA domains are found in several ubiquitin pathway proteins and in DNA repair enzymes such as Rad23, and they may be involved in ubiquitin binding. The presence of UBA domains provides a possible link between DNA methylation and ubiquitin/proteasome pathways.

A maize homolog of mammalian CENPC is a constitutive component of the inner kinetochore.

Genes for three maize homologs (CenpcA, CenpcB, and CenpcC) of the conserved kinetochore assembly protein known as centromere protein C (CENPC) have been identified. The C-terminal portion of maize CENPC shares similarity with mammalian CENPC and its yeast homolog Mif2p over a 23-amino acid region known as region I. Immunolocalization experiments combined with three-dimensional light microscopy demonstrated that CENPC is a component of the kinetochore throughout interphase, mitosis, and meiosis. It is shown that sister kinetochore separation occurs in two discrete phases during meiosis. A partial separation of sister kinetochores occurs in prometaphase I, and a complete separation occurs in prometaphase II. CENPC is absent on structures known as neocentromeres that, in maize, demonstrate poleward movement but lack other important features of centromeres/kinetochores. CENPC and a previously identified centromeric DNA sequence interact closely but do not strictly colocalize on meiotic chromosomes. These and other data indicate that CENPC occupies an inner domain of the maize kinetochore.

The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain.

The dihydrolipoamide S-acetyltransferase (E2) subunit of the maize mitochondrial pyruvate dehydrogenase complex (PDC) was postulated to contain a single lipoyl domain based upon molecular mass and N-terminal protein sequence (Thelen, J. J., Miernyk, J. A., and Randall, D. D. (1998) Plant Physiol. 116, 1443-1450). This sequence was used to identify a cDNA from a maize expressed sequence tag data base. The deduced amino acid sequence of the full-length cDNA was greater than 30% identical to other E2s and contained a single lipoyl domain. Mature maize E2 was expressed in Escherichia coli and purified to a specific activity of 191 units mg(-1). The purified recombinant protein had a native mass of approximately 2.7 MDa and assembled into a 29-nm pentagonal dodecahedron as visualized by electron microscopy. Immunoanalysis of mitochondrial proteins from various plants, using a monoclonal antibody against the maize E2, revealed 50-54-kDa cross-reacting polypeptides in all samples. A larger protein (76 kDa) was also recognized in an enriched pea mitochondrial PDC preparation, indicating two distinct E2s. The presence of a single lipoyl-domain E2 in Arabidopsis thaliana was confirmed by identifying a gene encoding a hypothetical protein with 62% amino acid identity to the maize homologue. These data suggest that all plant mitochondrial PDCs contain an E2 with a single lipoyl domain. Additionally, A. thaliana and other dicots possess a second E2, which contains two lipoyl domains and is only 33% identical at the amino acid level to the smaller isoform. The reason two distinct E2s exist in dicotyledon plants is uncertain, although the variability between these isoforms, particularly within the subunit-binding domain, suggests different roles in assembly and/or function of the plant mitochondrial PDC.

The maize homologue of the cell cycle checkpoint protein MAD2 reveals kinetochore substructure and contrasting mitotic and meiotic localization patterns.

We have identified a maize homologue of yeast MAD2, an essential component in the spindle checkpoint pathway that ensures metaphase is complete before anaphase begins. Combined immunolocalization of MAD2 and a recently cloned maize CENPC homologue indicates that MAD2 localizes to an outer domain of the prometaphase kinetochore. MAD2 staining was primarily observed on mitotic kinetochores that lacked attached microtubules; i.e., at prometaphase or when the microtubules were depolymerized with oryzalin. In contrast, the loss of MAD2 staining in meiosis was not correlated with initial microtubule attachment but was correlated with a measure of tension: the distance between homologous or sister kinetochores (in meiosis I and II, respectively). Further, the tension-sensitive 3F3/2 phosphoepitope colocalized, and was lost concomitantly, with MAD2 staining at the meiotic kinetochore. The mechanism of spindle assembly (discussed here with respect to maize mitosis and meiosis) is likely to affect the relative contributions of attachment and tension. We support the idea that MAD2 is attachment-sensitive and that tension stabilizes microtubule attachments.

Direct ultrastructural imaging of macrophages using a novel x-ray contact microscopy.

A compact, high-resolution, laser-plasma, x-ray contact microscopy method using a table-top Nd:glass laser system has been developed. This x-ray microscopy system was applied for the observation of macrophage ultrastructures. These images were produced using proximity imaging in which a 5-ns pulse of soft x-rays with wavelengths near and inside the water windows (23A-44A) produced by the laser-plasma were absorbed by the specimen and then registered on a photo resist. The x-ray images imprinted on the photo resist were then developed and analyzed with an atomic force microscope (AFM). Mouse thioglycollate-elicited peritoneal macrophages in suspension were examined by this new x-ray microscope. The x-ray images of the macrophages were compared with those observed by conventional transmission electron microscopy (TEM). The x-ray images showed no obvious organelles, including the nucleus and endoplasmic reticulum, as can be seen with TEM, but high- and low-contrast structures caused by mass distribution of carbon were observed. Thus, using the x-ray microscopy we visualized the first x-ray images of macrophage ultrastructures. The successful x-ray imaging of macrophage ultrastructure indicates that proximity x-ray microscopy may be of value in studying physiology linked to the dynamics of a cell.

Molecular analysis of two pyruvate dehydrogenase kinases from maize.

Two maize cDNAs were isolated and sequenced that had open reading frames with approximately 37% amino acid identity to mammalian pyruvate dehydrogenase kinases. Both maize kinase sequences contain the five domains with conserved signature residues typical of procaryotic two-component histidine kinases. Sequence comparisons identified six other highly conserved motifs that are proposed to be specific to pyruvate dehydrogenase kinases. In addition, specific Trp and Cys residues are also invariant in these sequences. The maize cDNAs are 1332 (PDK1) and 1602 (PDK2) nucleotides in length, encoding polypeptides with calculated molecular masses of 38,867 and 41,327 Da that share 77% amino acid identity. Reverse transcriptase-polymerase chain reaction analysis with oligonucleotide-specific primers revealed a differential expression pattern for the two isoforms. PDK1 and PDK2 were expressed in Escherichia coli with N-terminal His6 tags to facilitate purification. The recombinant proteins migrated at 44 and 48 kDa, respectively, during SDS-polyacrylamide gel electrophoresis. Anti-PDK1 antibodies immunoprecipitated 75% of pyruvate dehydrogenase kinase activity from a maize mitochondrial matrix fraction, and recognized a matrix protein of 43 kDa. Recombinant PDK2, expressed as a fusion with the maltose-binding protein, inactivated kinase-depleted maize pyruvate dehydrogenase complex when incubated with MgATP, coincident with incorporation of 32P from [gamma-32P]ATP into the alpha subunit of pyruvate dehydrogenase.

Surgical trauma evokes a rise in the frequency of hematopoietic progenitor cells and cytokine levels in blood circulation.

Alterations in the mononuclear cell populations in the blood circulation are among the most characteristic changes after surgical trauma. They reflect changes in the hematopoietic compartment which develop following surgery. The process of mobilization and differentiation of the hematopoietic population is regulated by cytokines known as growth factors for stem and progenitor cells (SCF, IL-1, IL-3, IL-6, IL-1L, TNF, CSFs). Our question was whether operative trauma resulted in the release of the hematopoietic progenitor cells to the blood circulation and an increase in the blood level of cytokines participating in hematopoiesis. The studies were carried out in patients with chronic cholelithiasis, undergoing elective open cholecystectomy under general anesthesia. An increase in the frequency of circulating CD34+ hematopoietic progenitor cells was seen between days 3 and 7 after surgery. Moreover, a significant increase in the percentage of immature cells of myeloid lineage (CD13+, CD14+, CD33+) was seen on the 1st and 3rd postoperative days. This could be the result of an expansion of the total bone marrow cell number after surgery and a subsequent release of these cells into the blood circulation. The changes in blood cell populations were accompanied by an increase in IL-6 on days 1, 3, and 7 following surgery, in IL-6sR on days 10 and 14 and in IL-8 on days 1 and 3. No significant changes in IL-1alpha, IL-1beta, IL-3 and IL-11 were noted. A small rise in GM-CSF was noted in few patients on the 3rd and 7th postoperative days. It is known that IL6 is involved in hematopoiesis, that the IL6-IL-6sR complex may induce both proliferation and differentiation of hematopoietic progenitor cells and that IL8 possesses progenitor cell mobilization properties. The appearance of hematopoietic progenitor cells in the blood following surgery may represent a process for the expansion of the immune cell pool after trauma and maintaining of the reserves at a certain level.

Application of X-ray micrography and imaging to study the effect of gentamicin on Pseudomonas aeruginosa.

Aminoglycosides disrupt the outer membrane of Pseudomonas aeruginosa to facilitate access to their intracellular target. High-resolution X-ray micrography of live specimens is a relatively new technique. We used laser (nanosecond) plasma to image live cells of P. aeruginosa ATCC 27853. After exposure to 25 mg/L gentamicin for 15 min. we observed perturbation of the cell surface, membrane blebbings (370 nm and 273 nm diameter) away from the cell, formation of distinct channels (241 nm long) resulting from indentation and induction of cell elongation from 3-3.6 microm (control) to 4.6-5.26 microm (gentamicin-treated cells). These data illustrate the potential of high-resolution X-ray micrography for studying effects of drugs on live microbiological specimens.

Sensitization of Burkholderia cepacia to antibiotics by cationic drugs.

Chlorpromazine and prochlorperazine have previously been shown to enhance the susceptibility of Burkholderia cepacia to aminoglycosides. To screen other non-antibiotic drugs containing similar amine (-N-CH3) groups, we examined a range of such agents that are in current clinical use for the treatment of non-infectious diseases, in combination with antibiotics that are ineffective against B. cepacia. At a concentration of 0.2 mM, theobromine, theophylline, trifluoperazine, fluophenazine and coumarin-152 significantly reduced (by four-fold) the MICs of gentamicin and ceftazidime. Theobromine and theophylline also reduced the MICs of amikacin and azithromycin.

Caspase-3-induced gelsolin fragmentation contributes to actin cytoskeletal collapse, nucleolysis, and apoptosis of vascular smooth muscle cells exposed to proinflammatory cytokines.

Gelsolin, an 80 kDa actin-severing protein, has been recently identified as a substrate for the cell death-promoting cysteinyl protease caspase-3 (CPP32/apopain/YAMA). We investigated the role of gelsolin and its cleavage product in apoptosis of vascular smooth muscle cells (SMC) induced by the proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Treatment with a combination of IFN-gamma and TNF-alpha reduced viability of SMC in a time- and concentration-dependent manner. Immunoblotting revealed that SMC treated with the cytokines generated a 41 kDa gelsolin fragment. The gelsolin fragmentation required activation of caspase-3, as the caspase-3 inhibitor diminished cytokine-induced cell death as well as the fragmentation. Gelsolin cleavage was accompanied by a reduction in F-actin content and by a marked disruption of cell structure. Adenovirus-mediated transfection of this N-terminal gelsolin fragment into SMC altered cell morphology, reduced cell viability, increased the number of TUNEL-positive cells, and promoted internucleosomal DNA fragmentation. Compared to wild-type cells, gelsolin-deficient SMC showed resistance to apoptosis induced by the inflammatory cytokines. These results suggest a mechanistic role for gelsolin cleavage during SMC apoptosis, a process implicated in vessel development as well as stability of atherosclerotic plaque.

Fas is expressed in human atherosclerotic intima and promotes apoptosis of cytokine-primed human vascular smooth muscle cells.

The membrane protein Fas/Apo-1/CD95 signals programmed cell death or apoptosis in activated T lymphocytes. Vascular smooth muscle cells (SMCs) bear markers of programmed cell death or apoptosis in advanced atherosclerotic plaques that contain immune cells e.g., macrophages and T lymphocytes. This study tested the hypothesis that the Fas death-signaling pathway contributes to apoptosis of SMCs exposed to proinflammatory cytokines produced by these immune cells during atherogenesis. All atherosclerotic plaques examined (n = 14) contained immunoreactive Fas. The majority of the Fas+ SMCs localized in the intima of the plaques, whereas the medial SMCs expressed Fas antigen less prominently. Double staining for DNA fragments (TUNEL) and Fas or cell identification markers colocalized Fas with TUNEL+ SMCs in the areas that contained CD3+ T cells and CD68+ macrophages, suggesting a role for Fas in the induction of SMC apoptosis by activated T cells during atherogenesis. In culture, stimulation with interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta increased expression of Fas in SMCs. Incubation with an activating anti-Fas antibody triggered apoptosis of the cytokine-primed but not the untreated SMCs, as demonstrated by TUNEL and electrophoresis of oligonucleosomal DNA fragments. These data suggest that activation of the Fas death-signaling pathway contributes to the induction of SMC apoptosis during atherogenesis and furnish a mechanism whereby immune cells and their cytokines promote this cell death process related to vascular remodeling and plaque rupture.

Enhancement of Burkholderia cepacia antimicrobial susceptibility by cationic compounds.

Infections in cystic fibrosis (CF) due to Burkholderia cepacia are challenging due to their resistance to antibiotics. We explored a new strategy for increasing the permeability of B. cepacia using cationic agents, including amino compounds, to reduce the MICs of standard antibiotics. Twenty-eight B. cepacia isolates from four CF centres in North America and four non-CF B. cepacia were examined by standard microtitre broth dilution methods for susceptibility to a variety of antibiotics in the presence of non-inhibitory concentrations of diaminoacetone (DAA), methylglyoxal bis-guanylhydrazone (MGBH), chlorpromazine (CPZ) and prochlorperazine (PCPZ). The proportion of isolates with greater than four-fold reductions in MIC in the presence of 0.3 mM CPZ or 0.4 mM PCPZ were 90% and 94% for gentamicin, 80% and 83% for tobramycin, 45% and 17% for ceftazidime, and 35% and 17% for amifloxacin. CPZ showed the same degree of reduction in the MIC of azithromycin in 79% strains (MIC50 reduced to 16 from > or = 256 mg/L). Non-CF B. cepacia showed a greater than four-fold reduction in MIC with CPZ for gentamicin, tobramycin and azithromycin and two-fold reduction for ceftazidime. Little or no reduction in MIC was seen with DAA or MGBH for any antibiotic. Addition of magnesium ions to the medium competitively inhibited any MIC reduction effect seen with the cationic agents. CPZ and PCPZ appeared to enhance the permeability of B. cepacia to antibiotics based upon ionic charge characteristics of the antibiotic. No significant differences were seen in outer membrane protein and lipopolysaccharide profiles between the culture treated with CPZ and the respective control culture of strain B. cepacia ATCC 13945. The fluorescent probe 1N-phenylnaphthylamine had no increased access across the outer membrane in the presence of CPZ for B. cepacia ATCC 13945. However, thin-section electron microscopy revealed separation between the outer membrane and the rest of the cytoplasm accompanied by a widening of the periplasmic space. These data provide a rationale for investigating amino compounds as potential permeability-increasing agents against B. cepacia.

X-ray micrography and imaging of Escherichia coli cell shape using laser plasma pulsed point x-ray sources.

High-resolution x-ray microscopy is a relatively new technique and is performed mostly at a few large synchrotron x-ray sources that use exposure times of seconds. We utilized a bench-top source of single-shot laser (ns) plasma to generate x-rays similar to synchrotron facilities. A 5 microlitres suspension of Escherichia coli ATCC 25922 in 0.9% phosphate buffered saline was placed on polymethylmethyacrylate coated photoresist, covered with a thin (100 nm) SiN window and positioned in a vacuum chamber close to the x-ray source. The emission spectrum was tuned for optimal absorption by carbon-rich material. Atomic force microscope scans provided a surface and topographical image of differential x-ray absorption corresponding to specimen properties. By using this technique we observed a distinct layer around whole cells, possibly representing the Gram-negative envelope, darker stained areas inside the cell corresponding to chromosomal DNA as seen by thin section electron microscopy, and dent(s) midway through one cell, and 1/3- and 2/3-lengths in another cell, possibly representing one or more division septa. This quick and high resolution with depth-of-field microscopy technique is unmatched to image live hydrated ultrastructure, and has much potential for application in the study of fragile biological specimens.

X-ray microscopy and imaging of Escherichia coli, LPS and DNA.

Ultrastructural examination by transmission and scanning electron microscopy involves a series of specialized preparation steps which may introduce artefacts in the micrographs. X-ray microscopy can take instant images of specimens but is mostly restricted to a few synchrotron X-ray sources. We have utilized a bench-top nanosecond laser-plasma to produce a single-shot source of nanosecond X-rays tuned for maximum contrast with carbon-rich material. To examine the ultrastructure by absorption profiles, we utilized a laser-produced plasma generated by a single-shot laser (1.06 microns wavelength, 5 x 10(12) W cm-2 intensity) focused on to a silicon target as an X-ray source for high-resolution X-ray microscopy. This approach eliminates the specimen preparation steps. Whole hydrated cells of Escherichia coli and purified preparations of lipopolysaccharide (LPS) and chromosomal DNA (cDNA) were streaked onto poly(methyl methacrylate) (PMMA)-coated grids (resist). This resist was exposed to X-rays under vacuum at a distance of 2.5 cm from the target disc. The silicon plasma produced by a 10-ns burst of laser energy (at 20J) radiates strong emission lines in the region of 300 eV. The X-rays penetrate the sample and their absorption profile is transferred on to the resist where PMMA acts as a negative to generate an image. By atomic force microscopy imaging of this photoresist we have visualized layers around cells of E.coli, darker areas inside the cell probably corresponding to cDNA, and preliminary images of LPS and DNA molecules. This technique has resolution at the 100 A level, produces images similar to the space-filling models of macromolecules and may be of great value in the study of the ultrastructure of hydrated live biological specimens.

Association of resistance to trimethoprim/sulphamethoxazole, chloramphenicol and quinolones with changes in major outer membrane proteins and lipopolysaccharide in Burkholderia cepacia.

Antibiotic treatment options for Burkholderia cepacia infection are limited because of high intrinsic resistance. The problem is complicated by development of cross-resistance between antibiotics of different classes. We isolated antibiotic-resistant mutants by stepwise exposure to chloramphenicol (Chlor) and to trimethoprim/sulphamethoxazole (T/S) for four B. cepacia strains: ATCC13945, Per (clinical isolate), Cas and D4 (environmental isolates). Chlor(r) mutants did not produce chloramphenicol acetyl-transferase. Cross-resistance, defined as greater than four-fold increase in MIC by microtitre dilution method, was consistently seen in both types of mutants. For chloramphenicol-resistant (Chlor[r]) and trimethoprim/sulphamethoxazole-resistant (Tr/Sr) mutants of B. cepacia ATCC13945 and Cas, no MIC change was seen for piperacillin, ceftazidime, rifampicin, gentamicin, tobramycin, polymyxin B or azithromycin. B. cepacia-Per and -D4 mutants showed cross-resistance to ceftazidime and to piperacillin. Comparison of outer membrane protein (OMP) profiles of B. cepacia and their mutants by SDS-PAGE revealed Tr/Sr) mutants to be deficient in a major OMP (molecular weight 39-47 kDa). Tr/Sr mutants also expressed additional OMPs not found in wild type strains at 75-77 kDa for B. cepacia-ATCC13945 and -Cas, and 20-21 kDa in B. cepacia-D4 and -Per. No OMP changes occurred in Chlor(r) mutants. Lipopolysaccharide (LPS) profiles of each type of mutant showed new high and low molecular weight LPS bands. Cross-resistance seems to be mediated by alterations in porin and LPS for Tr/Sr mutants, but only by LPS in Chlor(r) mutants.