Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli.

Processes for the biological removal of phosphate from wastewater rely on temporary manipulation of bacterial polyphosphate levels by phased environmental stimuli. In E. coli polyphosphate levels are controlled via the polyphosphate-synthesizing enzyme polyphosphate kinase (PPK1) and exopolyphosphatases (PPX and GPPA), and are temporarily enhanced by PPK1 overexpression and reduced by PPX overexpression. We hypothesised that partitioning PPK1 from cytoplasmic exopolyphosphatases would increase and stabilise E. coli polyphosphate levels. Partitioning was achieved by co-expression of E. coli PPK1 fused with a microcompartment-targeting sequence and an artificial operon of Citrobacter freundii bacterial microcompartment genes. Encapsulation of targeted PPK1 resulted in persistent phosphate uptake and stably increased cellular polyphosphate levels throughout cell growth and into the stationary phase, while PPK1 overexpression alone produced temporary polyphosphate increase and phosphate uptake. Targeted PPK1 increased polyphosphate in microcompartments 8-fold compared with non-targeted PPK1. Co-expression of PPX polyphosphatase with targeted PPK1 had little effect on elevated cellular polyphosphate levels because microcompartments retained polyphosphate. Co-expression of PPX with non-targeted PPK1 reduced cellular polyphosphate levels. Thus, subcellular compartmentalisation of a polymerising enzyme sequesters metabolic products from competing catabolism by preventing catabolic enzyme access. Specific application of this process to polyphosphate is of potential application for biological phosphate removal.

Lysozyme and bilirubin bind to ACE and regulate its conformation and shedding.

Angiotensin I-converting enzyme (ACE) hydrolyzes numerous peptides and is a critical participant in blood pressure regulation and vascular remodeling. Elevated tissue ACE levels are associated with increased risk for cardiovascular and respiratory disorders. Blood ACE concentrations are determined by proteolytic cleavage of ACE from the endothelial cell surface, a process that remains incompletely understood. In this study, we identified a novel ACE gene mutation (Arg532Trp substitution in the N domain of somatic ACE) that increases blood ACE activity 7-fold and interrogated the mechanism by which this mutation significantly increases blood ACE levels. We hypothesized that this ACE mutation disrupts the binding site for blood components which may stabilize ACE conformation and diminish ACE shedding. We identified the ACE-binding protein in the blood as lysozyme and also a Low Molecular Weight (LMW) ACE effector, bilirubin, which act in concert to regulate ACE conformation and thereby influence ACE shedding. These results provide mechanistic insight into the elevated blood level of ACE observed in patients on ACE inhibitor therapy and elevated blood lysozyme and ACE levels in sarcoidosis patients.

Visualization of the glomerular endothelial glycocalyx by electron microscopy using cationic colloidal thorium dioxide.

Biological material itself appears with poor contrast in electron microscopy (EM), due to its composition mostly of light elements. Classical staining agents such as osmium tetroxide, uranyl acetate, and lead citrate preserve and/or stain cellular structures such as membranes, cytoplasm, and organelles well for EM. However, extracellular polymeric substances (EPS) show no or only poor contrast with these staining agents. The endothelial glycocalyx in blood vessels consists mainly of proteoglycans. It can be visualized by EM only by additional staining with heavy metal ions such as copper (Alcian blue, cupromeronic blue), ruthenium (ruthenium red), or lanthanum. Best results are achieved by combined perfusion of fixative and stain. Cationic hydrous thorium dioxide colloids (named here cThO2) trace acidic groups in EPS. We describe here the use of cThO2 to visualize the glomerular endothelial glycocalyx in the mouse kidney. cThO2 shows high electron density and binds to a continuous layer of up to a few hundred nanometers thickness on the glomerular endothelium, as well as on epithelia in other blood vessels in perfused animals. The observed staining pattern gives rise to periodic densities, with a spacing varying between 50 and 200 nm, depending on the overall layer thickness, which varies between below 50 up to 300 nm. Due to high electron density of the used cThO2 particles, the introduced method allows distinct imaging and precise fine structural analysis of the endothelial glycocalyx.

Isoniazid@Fe2 O3 Nanocontainers and Their Antibacterial Effect on Tuberculosis Mycobacteria.

Isoniazid-filled Fe2 O3 hollow nanospheres (INH@Fe2 O3 , diameter <30 nm, 48 wt % INH-load) are prepared for the first time and suggested for tuberculosis therapy. After dextran-functionalization, the INH@Fe2 O3 @DEX nanocontainers show strong activity against Mycobacterium tuberculosis (M.tb.) and M.tb.-infected macrophages. The nanocontainers can be considered as "Trojan horses" and show efficient, active uptake into both M.tb.-infected macrophages and even into mycobacterial cells.

The 3D structure of Kaposi sarcoma herpesvirus LANA C-terminal domain bound to DNA.

Kaposi sarcoma herpesvirus (KSHV) persists as a latent nuclear episome in dividing host cells. This episome is tethered to host chromatin to ensure proper segregation during mitosis. For duplication of the latent genome, the cellular replication machinery is recruited. Both of these functions rely on the constitutively expressed latency-associated nuclear antigen (LANA) of the virus. Here, we report the crystal structure of the KSHV LANA DNA-binding domain (DBD) in complex with its high-affinity viral target DNA, LANA binding site 1 (LBS1), at 2.9 Šresolution. In contrast to homologous proteins such as Epstein-Barr virus nuclear antigen 1 (EBNA-1) of the related γ-herpesvirus Epstein-Barr virus, specific DNA recognition by LANA is highly asymmetric. In addition to solving the crystal structure, we found that apart from the two known LANA binding sites, LBS1 and LBS2, LANA also binds to a novel site, denoted LBS3. All three sites are located in a region of the KSHV terminal repeat subunit previously recognized as a minimal replicator. Moreover, we show that the LANA DBD can coat DNA of arbitrary sequence by virtue of a characteristic lysine patch, which is absent in EBNA-1 of the Epstein-Barr virus. Likely, these higher-order assemblies involve the self-association of LANA into supermolecular spirals. One such spiral assembly was solved as a crystal structure of 3.7 Šresolution in the absence of DNA. On the basis of our data, we propose a model for the controlled nucleation of higher-order LANA oligomers that might contribute to the characteristic subnuclear KSHV microdomains ("LANA speckles"), a hallmark of KSHV latency.

Contribution of Veillonella parvula to Pseudomonas aeruginosa-mediated pathogenicity in a murine tumor model system.

The recent finding that high numbers of strict anaerobes are present in the respiratory tract of cystic fibrosis (CF) patients has drawn attention to the pathogenic contribution of the CF microbiome to airway disease. In this study, we investigated the specific interactions of the most dominant bacterial CF pathogen, Pseudomonas aeruginosa, with the anaerobic bacterium Veillonella parvula, which has been recovered at comparable cell numbers from the respiratory tract of CF patients. In addition to growth competition experiments, transcriptional profiling, and analyses of biofilm formation by in vitro studies, we used our recently established in vivo murine tumor model to investigate mutual influences of the two pathogens during a biofilm-associated infection process. We found that P. aeruginosa and V. parvula colonized distinct niches within the tumor. Interestingly, significantly higher cell numbers of P. aeruginosa could be recovered from the tumor tissue when mice were coinfected with both bacterial species than when mice were monoinfected with P. aeruginosa. Concordantly, the results of in vivo transcriptional profiling implied that the presence of V. parvula supports P. aeruginosa growth at the site of infection in the host, and the higher P. aeruginosa load correlated with clinical deterioration of the host. Although many challenges must be overcome to dissect the specific interactions of coinfecting bacteria during an infection process, our findings exemplarily demonstrate that the complex interrelations between coinfecting microorganisms and the immune responses determine clinical outcome to a much greater extent than previously anticipated.

Highly stable monodisperse PEGylated iron oxide nanoparticle aqueous suspensions: a nontoxic tracer for homogeneous magnetic bioassays.

Uniformly sized and shaped iron oxide nanoparticles with a mean size of 25 nm were synthesized via decomposition of iron-oleate. High resolution transmission electron microscopy and Mössbauer spectroscopy investigations revealed that the particles are spheres primarily composed of Fe3O4 with a small fraction of FeO. From Mössbauer and static magnetization measurements, it was deduced that the particles are superparamagnetic at room temperature. The hydrophobic particles were successfully transferred into water via PEGylation using nitrodopamine as an anchoring group. IR spectroscopy and thermogravimetric analysis showed the success and efficiency of the phase transfer reaction. After PEGylation, the particles retained monodispersity and their magnetic core remained intact as proven by photon cross-correlation spectroscopy, ac susceptibility, and transmission electron microscopy. The particle aqueous suspensions revealed excellent water stability over a month of monitoring and also against temperature up to 40 °C. The particles exhibited a moderate cytotoxic effect on in vitro cultured bone marrow-derived macrophages and no release of inflammatory or anti-inflammatory cytokines. The PEGylated particles were functionalized with Herceptin antibodies via a conjugation chemistry, their response to a rotating magnetic field was studied using a fluxgate-based setup and was compared with the one recorded for hydrophobic and PEGylated particles. The particle phase lag rose after labeling with Herceptin, indicating the successful conjugation of Herceptin antibodies to the particles.

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.

Dengue-specific subviral nanoparticles: design, creation and characterization.

BACKGROUND: Dengue is today the most significant of arboviral diseases. Novel tools are necessary to effectively address the problem of dengue. Virus-like particles (VLP) offer a versatile nanoscale platform for developing tools with potential biomedical applications. From the perspective of a potentially useful dengue-specific tool, the dengue virus envelope protein domain III (EDIII), endowed with serotype-specificity, host receptor recognition and the capacity to elicit virus-neutralizing antibodies, is an attractive candidate. METHODS: We have developed a strategy to co-express and co-purify Hepatitis B virus surface (S) antigen in two forms: independently and as a fusion with EDIII. We characterized these physically and functionally. RESULTS: The two forms of the S antigen associate into VLPs. The ability of these to display EDIII in a functionally accessible manner is dependent upon the relative levels of the two forms of the S antigen. Mosaic VLPs containing the fused and un-fused components in 1:4 ratio displayed maximal functional competence. CONCLUSIONS: VLPs armed with EDIII may be potentially useful in diagnostic, therapeutic and prophylactic applications.

Cauliform bacteria lacking phospholipids from an abyssal hydrothermal vent: proposal of Glycocaulis abyssi gen. nov., sp. nov., belonging to the family Hyphomonadaceae.

Cauliform bacteria are prosthecate bacteria often specialized for oligotrophic environments. A polyphasic approach, comprising 16S rRNA gene sequencing, lipid analysis and salt tolerance characterizations, was used to clarify the taxonomy of one isolate, strain MCS 33(T), obtained from above the hot water plume of a deep-sea hydrothermal vent near Vancouver island, Canada. Cells contained no detectable phospholipids or sulpholipids, but did contain 1,2-di-O-acyl-3-O-α-D-glucopyranosylglycerol, 1,2-di-O-acyl-3-O-α-D-glucopyranuronosylglycerol and the novel lipid 1,2-di-O-acyl-3-[O-α-D-glucopyranuronosyl]glycerol-6'-N-glycine. It is assumed that the various glucoronosyl lipids are replacing, at least partially, the phospholipids in their various tasks in the cell cycle. The G+C content of the genomic DNA of strain MCS 33(T) was 62.8 mol%, and Q10 was the predominant respiratory ubiquinone. The 16S rRNA gene sequence of this chemoheterotrophic, aerobic, moderately halophilic strain showed only a low similarity of 94.4% to that of Oceanicaulis alexandrii C116-18(T), and both strains also differed based on their lipids. Although the novel strain was isolated from seawater sampled near a hydrothermal vent, its optimum temperature for growth was 30 °C. The main cellular fatty acids were C18:1ω7c, C18:0 and the unknown fatty acid ECL 11.798, and the main hydroxy fatty acid was C12:0 3-OH. The strain is proposed to represent a novel species of a new genus, Glycocaulis abyssi gen. nov., sp. nov. The type strain of the type species is MCS 33(T) (=LMG 27140(T)=CCUG 62981(T)).

Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the hepatitis B surface antigen.

BACKGROUND: A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification. RESULTS: High level production of HBsAg was carried out with recombinant Pichia pastoris using the methanol inducible AOX1 expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell. CONCLUSIONS: It is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were not found within the cells it is concluded that the VLP assembly process must take place during down-stream processing after detergent-mediated disassembly of HBsAg lamellas and subsequent reassembly of HBsAg into spherical VLPs.

Transcriptional profiling of the marine oil-degrading bacterium Alcanivorax borkumensis during growth on n-alkanes.

The marine oil-degrading bacterium Alcanivorax borkumensis SK2 has attracted significant interest due to its hydrocarbonoclastic lifestyle, its alkane-centered metabolism, and for playing an important ecological role in cleaning up marine oil spills. In this study, we used microarray technology to characterize the transcriptional responses of A. borkumensis to n-hexadecane exposure as opposed to pyruvate, which led to the identification of a total of 220 differentially expressed genes, with 109 genes being upregulated and 111 genes being downregulated. Among the genes upregulated on alkanes are systems predicted to be involved in the terminal oxidation of alkanes, biofilm formation, signal transduction, and regulation.

Gordonibacter pamelaeae gen. nov., sp. nov., a new member of the Coriobacteriaceae isolated from a patient with Crohn's disease, and reclassification of Eggerthella hongkongensis Lau et al. 2006 as Paraeggerthella hongkongensis gen. nov., comb. nov.

A strictly anaerobic, Gram-positive, short-rod/coccobacillus-shaped bacterial strain, designated 7-10-1-b(T), was isolated from the colon of a patient suffering from acute Crohn's disease. The isolate formed small, pale-white, semi-translucent colonies on solid cultivation media. The strain was catalase-positive and metabolized only a small number of carbon sources. Whole-cell fatty acids consisted predominantly of saturated fatty acids (89 %), of which 15 : 0 anteiso was the major component. The polar lipids phosphatidylglycerol and diphosphatidylglycerol as well as four glycolipids were identified. 16S rRNA gene sequence analysis revealed that the isolate represents a distinct lineage within the family Coriobacteriaceae and has 94.6 % identity to the type strain of [Eggerthella] hongkongensis, the phylogenetically closest bacterial species. On the basis of the analyses performed, the new genus and species Gordonibacter pamelaeae gen. nov., sp. nov. is described, with strain 7-10-1-b(T) (=DSM 19378(T) =CCUG 55131(T)) as the type and only strain of Gordonibacter pamelaeae. Also, based on the chemotaxonomic data obtained for all type strains of the neighbouring genus Eggerthella, we propose that Eggerthella hongkongensis Lau et al. 2006 be transferred to a new genus as Paraeggerthella hongkongensis gen. nov., comb. nov.; the type strain of Paraeggerthella hongkongensis is HKU10(T) (=DSM 16106(T) =CCUG 49250(T)).

Phylogeny by a polyphasic approach of the order Caulobacterales, proposal of Caulobacter mirabilis sp. nov., Phenylobacterium haematophilum sp. nov. and Phenylobacterium conjunctum sp. nov., and emendation of the genus Phenylobacterium.

Three strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from fresh water and human blood. As determined by analyses of 16S rRNA gene sequences, the prosthecate strain FWC 38T was affiliated to the alphaproteobacterial genus Caulobacter, with Caulobacter henricii (96.8 %) and Caulobacter fusiformis (96.8 %) as its closest relatives. The non-prosthecate strain LMG 11050T and the prosthecate strain FWC 21T both belonged to the genus Phenylobacterium with Phenylobacterium koreense (96.9 %) and Phenylobacterium immobile (96.3 %) as the closest relatives. This affiliation was supported by chemotaxonomic data (polar lipids and cellular fatty acids). Physiological and biochemical tests allowed genotypic and phenotypic differentiation of the novel strains from all hitherto recognized species of the genera Caulobacter and Phenylobacterium. The strains therefore represent novel species, for which the names Caulobacter mirabilis sp. nov. (type strain FWC 38T=LMG 24261T=CCUG 55073T), Phenylobacterium conjunctum (type strain FWC 21T=LMG 24262T=CCUG 55074T), the first described prosthecate Phenylobacterium species, and Phenylobacterium haematophilum sp. nov. (type strain LMG 11050T=CCUG 26751T) are proposed. Marker nucleotides within the 16S rRNA genes were determined for the genera Asticcacaulis, Brevundimonas, Caulobacter and Phenylobacterium and the description of the genus Phenylobacterium is emended.

Biostraticola tofi gen. nov., spec. nov., a novel member of the family Enterobacteriaceae.

Bacterial strain BF36T, isolated from the biofilm of a tufa deposit in a hard water rivulet, was characterized by a polyphasic taxonomic approach. Cells of these organisms were Gram-negative, motile, nonpigmented, rod-shaped, non-endospore-forming, and facultatively anaerobic. Cells, organized in loose consortia, were coated by a massive slime layer. Phylogenetic analyses using 16S rRNA gene sequences showed that strain BF36T was a member of the family Enterobacteriaceae, class Gammaproteobacteria, displaying a moderate degree of relationship (96.5% sequence similarity) to Sodalis glossinidius and "Sodalis pallipedes," intracellular symbionts of the tsetse fly Glossinis morsitans morsitans. Dendrograms of relationship generated by different algorithms consistently grouped isolate BF36T with Sodalis glossinidius, Pragia fontium, Budvicia aquatica, Serratia rubideae, and Brenneria spp (94.7-95.8% similarity) which also share many common metabolic properties. Differences between strain BF36T and Sodalis glossinidius DSM 13495T are seen in motility and in the pattern of substrates utilized. Membership to the family was also confirmed by a fatty acid profile consisting of major amounts of C16:0)and C16:1omega7, by the presence of isoprenoids of the ubiquinone Q8 and menaquinone MK8 types and a DNA G + C content of 54.2 mol%. The decision to classify strain BF36T into a new genus Biostraticola gen. nov. is based on its distant phylogenetic position as compared to any other representative of the family and the significant phenotypic differences to its nearest phylogenetic neighbor, Sodalis glossinidius. BF36T represents the type species, for which the name Biostraticola tofi sp. nov. is proposed. The type strain is BF36T (DSM 19580T; CIP109699T).

Deefgea rivuli gen. nov., sp. nov., a member of the class Betaproteobacteria.

Two strains, designated WB 3.4-79(T) and WB 3.3-25, were isolated from a hard-water sample collected from the Westerhöfer Bach, Lower Saxony, Germany. The strains shared 100 % DNA-DNA relatedness, indicating membership of the same genospecies. This close relationship was supported by identical 16S rRNA gene sequences and high similarities in fatty acid composition and biochemical characteristics. The G+C content of the genomic DNA of strain WB 3.4-79(T) was 48.5 mol% and the predominant ubiquinone was Q-8. Major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. Major fatty acids (>10 %) were C(16 : 0) and C(16 : 1)omega7c. Polyhydroxybutyrate and polyphosphate granules as well as unidentified enterosomes and a polar organelle are visible by electron microscopy. Comparative 16S rRNA gene sequence analysis indicated that the isolates were placed within the class Betaproteobacteria, remotely related to Chitinibacter tainanensis DSM 15459(T), Silvimonas terrae KCTC 12358(T), Formivibrio citricus DSM 6150(T) and Iodobacter fluviatilis DSM 3764(T). On the basis of phylogenetic and phenotypic distinctness, we propose a novel genus, Deefgea gen. nov., with Deefgea rivuli sp. nov. as the type species. The type strain of Deefgea rivuli is strain WB 3.4-79(T) (=DSM 18356(T)=CIP 109326(T)).

Cationic hydrous thorium dioxide colloids--a useful tool for staining negatively charged surface matrices of bacteria for use in energy-filtered transmission electron microscopy.

BACKGROUND: Synthesis of cationic hydrous thorium dioxide colloids (ca. 1.0 to 1.7 nm) has been originally described by Müller 22 and Groot 11 and these have been used by Groot to stain acidic glucosaminoglycans for ultrastructure research of different tissues by conventional transmission electron microscopy. RESULTS: Synthesis of colloidal thorium dioxide has been modified and its use as a suitable stain of acidic mucopolysaccharides and other anionic biopolymers from bacteria, either as whole mount preparations or as preembedment labels, is described. The differences in stain behavior relative to commonly used rutheniumred-lysine and Alcian Blue electron dense acidic stains has been investigated and its use is exemplified for Pseudomonas aeruginosa adjacent cell wall biopolymers. For the first time thorificated biopolymers, i.e. bacterial outer cell wall layers, have been analysed at the ultrastructural level with electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI), leading to excellent contrast and signal strength for these extracellular biopolymers. CONCLUSION: Application of cationic hydrous ThO2 colloids for tracing acidic groups of the bacterial surface and/or EPS has been shown to be rather effective by transmission electron microscopy. Because of its high electron density and its good diffusibility it stains and outlines electro-negative charges within these biopolymers. In combination with ESI, based on integrated energy-filtered electron microscopy (EFTEM) Th-densities and thus negative charge densities can be discriminated from other elemental densities, especially in environmental samples, such as biofilms.

Mechanism of action of tubulysin, an antimitotic peptide from myxobacteria.

Tubulysin A is a highly cytotoxic peptide with antimitotic activity that induces depletion of cell microtubules and triggers the apoptotic process. Treated cells accumulated in the G2/M phase. Tubulysin A inhibited tubulin polymerization more efficiently than vinblastine and induced depolymerization of isolated microtubule preparations. Microtubule depolymerization could not be prevented by preincubation with epothilone B and paclitaxel, neither in cell-free systems nor in cell lines. In competition experiments, tubulysin A strongly interfered with the binding of vinblastine to tubulin in a noncompetitive way; the apparent Ki was 3 microM. Electron microscopy investigations showed that tubulysin A induced the formation of rings, double rings, and pinwheel structures. The mode of action of tubulysin A resembled that of peptide antimitotics dolastatin 10, phomopsin A, and hemiasterlin. Efforts are underway to develop this new group of compounds as anticancer drugs.

Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung.

Pseudomonas aeruginosa colonizing the lung of cystic fibrosis patients is responsible for a decline in health and poor prognosis for these patients. Once established, growth of P. aeruginosa in microcolonies makes it very difficult to eradicate the organisms by antimicrobial treatment. An artificial sputum medium was developed to mimic growth of P. aeruginosa in the cystic fibrosis lung habitat and it was found that the organisms grew in tight microcolonies attached to sputum components. Several genes, such as algD, oprF and lasR but not fliC, were required for tight microcolony formation. Among the sputum components, amino acids, lecithin, DNA, salt and low iron were required for tight microcolony formation. Amino acids were also shown to be responsible for various other cystic-fibrosis-specific phenotypes of P. aeruginosa, such as diversification of colony morphology, alterations in LPS structure and hyperexpression of OprF. Since the amino acid content of sputum is elevated in severe lung disease, it is suggested that the tight microcolony biofilm is maintained in these conditions and that they contribute to the vicious cycle of disease severity and failure to eradicate the organism. Thus, growth of P. aeruginosa in artificial sputum medium is an appropriate model of chronic lung colonization and may be useful for evaluating therapeutic procedures and studying antibiotic-resistance mechanisms.

Growth of polychlorinated-biphenyl-degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxidative stress and massive accumulation of inorganic polyphosphate.

Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. Using transmission electron microscopy of the polychlorinated biphenyl (PCB)-degrading bacterium Pseudomonas sp. strain B4 grown in defined medium with biphenyl as the sole carbon source, we observed large and abundant electron-dense granules at all stages of growth and following a shift from glucose to biphenyl or chlorobiphenyls. Using energy dispersive X-ray analysis and electron energy loss spectroscopy with an integrated energy-filtered transmission electron microscope, we demonstrated that these granules were mainly composed of phosphate. Using sensitive enzymatic methods to quantify cellular polyP, we confirmed that this polymer accumulates in PCB-degrading bacteria when they grow in the presence of biphenyl and chlorobiphenyls. Concomitant increases in the levels of the general stress protein GroEl and reactive oxygen species were also observed in chlorobiphenyl-grown cells, indicating that these bacteria adjust their physiology with a stress response when they are confronted with compounds that serve as carbon and energy sources and at the same time are chemical stressors.