Cytologic manifestation of an unusual bacterial form, Simonsiella species

Puzzling rodlike structures overlying benign squamous cells in exfoliative cytologic specimens from the upper gastrointestinal and respiratory tracts were initially considered to be fungi, protozoa, bronchodilator crystals, hemoglobin tactoids or plastic fragments. Their morphologic similarity to Simonsiella, a gram-negative bacteria frequently found in the oral cavity, was ultimately recognized. Further studies of smears and cultures obtained from the oral cavities of the authors and from the American Type Culture Collection confirmed the nature of the original findings. These giant bacterial forms were usually found in caterpillarlike side-by-side arrangements of 10 to 12 organisms. Cytologists should be aware of their appearance to avoid possible confusion with pathogenic organisms.     

Understanding the binding properties of an unusual metal-binding protein--a study of bacterial frataxin

Deficiency of the small mitochondrial protein frataxin causes Friedreich's ataxia, a severe neurodegenerative pathology. Frataxin, which has been highly conserved throughout evolution, is thought to be involved in, among other processes, Fe-S cluster formation. Independent evidence shows that it binds iron directly, although with very distinct features and low affinity. Here, we have carried out an extensive study of the binding properties of CyaY, the bacterial ortholog of frataxin, to different divalent and trivalent cations, using NMR and X-ray crystallography. We demonstrate that the protein has low cation specificity and contains multiple binding sites able to chelate divalent and trivalent metals with low affinity. Binding does not involve cavities or pockets, but exposed glutamates and aspartates, which are residues that are unusual for iron chelation when not assisted by histidines and/or cysteines. We have related how such an ability to bind cations on a relatively large area through an electrostatic mechanism could be a valuable asset for protein function.     

Transposition of ISHp608, member of an unusual family of bacterial insertion sequences

ISHp608 from Helicobacter pylori is active in Escherichia coli and represents a recently recognised group of insertion sequences. Its transposase and organisation suggest that it transposes using a different mechanism to that of other known transposons. The IS was shown to excise as a circular form, which is accompanied by the formation of a resealed donor plasmid backbone. We also demonstrate that TnpA, which is less than half the length of other transposases, is responsible for this and for ISHp608 transposition. Transposition was shown to be site specific: both insertion and transposon excision require a conserved target, 5'TTAC. Deletion analysis suggested that potential secondary structures at the left and right ends are important for transposition. In vitro TnpA bound both ends, showed a strong preference for a specific single-stranded DNA and introduced a single-strand break on the same strand at each end. Although many of the characteristics of ISHp608 appear similar to rolling-circle transposons, there are differences suggesting that, overall, transposition occurs by a different mechanism. The results have permitted the formulation of several related models.     

A new method of preparation of pyocyanin and demonstration of an unusual bacterial sensitivity.

Pyocyanin was prepared in 60% yield from phenazine methoxysulfate by a photooxidation procedure and purification by silica gel chromatography. Monitoring was performed by thin-layer chromatography. Approximately 50% of clinical Pseudomonas aeruginosa isolates were found to produce pyocyanin at 37°C. Among Proteus strains, P. morganii strains were sensitive to concentrations of pyocyanin 16 to 64 times lower than concentrations that inhibited the growth of P. mirabilis and P. vulgaris strains.     

Life in an unusual intracellular niche: a bacterial symbiont infecting the nucleus of amoebae

Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named ‘Candidatus Nucleicultrix amoebiphila'', is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. ‘Candidatus Nucleicultrix amoebiphila'' traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96–120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations.     

Fatty acid desaturation: variations on an oxidative theme

Significant progress in our understanding of the mechanism of fatty acid desaturation has been achieved. The site of initial oxidation has been determined for several membrane-bound desaturases and a common cryptoregiochemical theme has been revealed. The results of several studies, including a detailed analysis of a soluble plant desaturase system, point to a close mechanistic relationship between dehydrogenation and hydroxylation pathways.     

外科手术切口感染病原菌的耐药性分析

目的探讨外科患者手术切口感染的病原菌分布及其耐药性。方法对158例外科住院患者手术后切口感染病原菌的分布及耐药性进行回顾性调查。结果培养出致病菌158株．细菌分布依次为金黄色葡萄球菌、铜绿假单胞菌、大肠埃希菌、凝固酶阴性葡萄球菌和肺炎克雷伯菌;其中分离出产ESBLs菌10株．耐亚胺培南菌6株,MRSA菌8株和MRCNS菌4株。耐药分析显示．万古霉素和亚胺培南仍具有较好的抗菌效果。结论应密切监测外科切口感染情况,合理用药,以减少感染发生。     

Production of unusual bacterial polyesters by Pseudomonas oleovorans through cometabolism

Abstract Pseudomonas oleovorans is an adaptable, aerobic bacterium that can produce a wide range of storage polyesters (poly-β-hydroxyalkanoates, PHAs). With few exceptions, the PHAs obtained when this bacterium is grown with single organic substrates capable of polymer production are generally copolymers. With two different polymer-producing substrates the copolymers formed contain units derived from each substrate often in direct proportion to the amounts in the medium. With such substrates or with non-producing substrates, or with non-growth substrates, the ability of P. oleovorans to utilize different types of individual organic compounds can be classified into three different categories, as follows: group A—the organic compound can support both growth and polymer production; or group B—the organic compound can support growth but not polymer production; or group C—the organic compounds cannot support growth. For organic compounds in groups B and C, new and unusual copolymers containing units derived from these substrates can often be obtained if that compound is cofed with a good polymer-producing substrate for P. oleovorans , such as either octanoic acid or nonanoic acid. The PHAs obtained by this type of cometabolism from a variety of such substrates are described.     

Structure and function of an unusual family of protein phosphatases: the bacterial chemotaxis proteins CheC and CheX

In bacterial chemotaxis, phosphorylated CheY levels control the sense of flagella rotation and thereby determine swimming behavior. In E. coli, CheY dephosphorylation by CheZ extinguishes the switching signal. But, instead of CheZ, many chemotactic bacteria contain CheC, CheD, and/or CheX. The crystal structures of T. maritima CheC and CheX reveal a common fold unlike that of any other known protein. Unlike CheC, CheX dimerizes via a continuous beta sheet between subunits. T. maritima CheC, as well as CheX, dephosphorylate CheY, although CheC requires binding of CheD to achieve the activity of CheX. Structural analyses identified one conserved active site in CheX and two in CheC; mutations therein reduce CheY-phosphatase activity, but only mutants of two invariant asparagine residues are completely inactive even in the presence of CheD. Our structures indicate that the flagellar switch components FliY and FliM resemble CheC more closely than CheX, but attribute phosphatase activity only to FliY.     

Benzene analysis in workplace air using an FIA-based bacterial biosensor

A bacterial biosensor based on flow injection analysis (FIA) has been developed for the determination of benzene in workplace air samples. Benzene can be used by the bacteria Pseudomonas putida ML2 as a sole carbon source, and its aerobic degradation can be measured using a dissolved oxygen electrode. The bacterial cells were immobilised between two cellulose acetate membranes and fixed onto a Clark dissolved oxygen probe, which was inserted into a custom-made flow cell. The applicability of the biosensor for the analysis of air samples containing benzene was investigated. Air samples were collected from a controlled exposure room using charcoal adsorption tubes, and benzene extracted with solvent desorption using dimethylformamide (DMF). The biosensor displayed a linear detection range between 0.025 and 0.15 mM benzene based on standard solutions containing a maximum of 2% DMF, with a response time of 6 min. This linear detection range allows the analysis of air containing between 3 and 16 ppm benzene based on a 60-min sampling period. DMF proved to be compatible for use with the biosensor, causing minimal interference with the sensor response and causing no toxic effects on the bacterial cells. The FIA system was easily transported to an in situ location, and a correlation was obtained between the biosensor and gas chromatography (GC) results for the preliminary air samples investigated. Moreover, the biosensor displayed no interference to other benzene related compounds in the BTEX range. The results from this work have shown that the biosensor has potential applications for the analysis of benzene in workplace air samples, with the added advantages over the conventional GC methods of low operation costs, ease of use, and portability for in situ measurements.     

Molecular analysis of a bacterial chitinolytic community in an upland pasture

The effects of agricultural-improvement treatments on the chitinolytic activity and diversity of a microbial community were investigated within an upland pasture. The treatments of interest were lime and treated sewage sludge, both commonly applied to pasture land to improve fertility. Burial of chitin-containing litter bags at the field site resulted in enrichment of bacteria according to 16S rRNA fingerprinting. Chitinolytic-activity measurements showed that the highest activity occurred in those bags recovered from sludge-amended plots, which correlated well with increased counts of actinobacteria in samples from these chitin bags. Our findings suggest that sewage sludge increases the fertility of the soil in terms of chitinase activity. Ten clone libraries were constructed from family 18 subgroup A chitinases, PCR amplified from litter bags buried in soil in July 2000 or in September 2000, in a separate study. Analysis of these libraries by restriction fragment length polymorphism and sequencing showed that they were dominated by actinobacterium-like chitinase sequences. This suggests that actinobacteria have an important chitinolytic function in this soil ecosystem. Our findings showed that sludge application increased chitinolytic activity but decreased the diversity of chitinases present.     

Crystallization of Photobacterium leiognathi non-fluorescent flavoprotein, an unusual flavoprotein with limited sequence identity to bacterial luciferase.

Single crystals of the non-fluorescent flavoprotein (NFP) purified from Photobacterium leiognathi strain S1 have been grown from ammonium sulphate solutions using the hanging drop vapour diffusion technique. The crystals grow as thin (0.06 mm) plates and belong to the orthorhombic space group C222(1): a = 57.06(3) A, b = 92.41(6) A, c = 99.52(6) A. There is one NFP monomer per asymmetric unit and crystals diffract to 2.2 A spacings on film. A complete native data set to 2.5 A resolution has been collected on a San Diego Multiwire Detector system at the University of Alberta and a heavy-atom derivative search is presently in progress.     

Sequence analysis of bacterial DNA in the colon of an Andean mummy

We have isolated DNA from 14 tissue samples from the internal organs of an Andean human mummy (10th–11th century A.D.) and have checked the persistence of the original human and bacterial templates using the following main approaches: 1) amino acid racemization test; 2) quantification of mitochondrial DNA copy number; 3) survey of bacterial DNA in the different organs; 4) sequence analysis of bacterial amplicons of different lengths. The results demonstrate that both the original human DNA and the DNA of the bacteria of the mummy gut are preserved. In particular, sequence analysis of two (respectively 100 and 196 bp in length) libraries of bacterial 16s ribosomal RNA gene amplicons from the mummy colon shows that while the shortest amplicons give only modest and biased indications about the bacterial taxa, the longer amplicons allow the identification several species of the genus Clostridium which are typical of the human colon. This work represents a first example of a methodological approach which is applicable, in principle, to many other natural and artificial mummies and might open the way to the study of the structure of the human microbial ecosystem from prehistory to present. Am J Phys Anthropol 107:285–295, 1998. © 1998 Wiley-Liss, Inc.     

New insights into the structure and oligomeric state of the bacterial multidrug transporter EmrE: an unusual asymmetric homo-dimer

EmrE is a small multidrug transporter that contains 110 amino acid residues that form four transmembrane alpha-helices. The three-dimensional structure of EmrE has been determined from two-dimensional crystals by electron cryo-microscopy. EmrE is an asymmetric homo-dimer with one substrate molecule bound in a chamber accessible laterally from one leaflet of the lipid bilayer. Evidence from substrate binding analyses and analytical ultracentrifugation of detergent-solubilised EmrE shows that the minimum functional unit for substrate binding is a dimer. However, it is possible that EmrE exists as a tetramer in vivo and plausible models are suggested based upon analyses of two-dimensional crystals.