Showing papers in "Microbiology in 2000"

Quantification of biofilm structures by the novel computer program COMSTAT.

Abstract: The structural organization of four microbial communities was analysed by a novel computer program, COMSTAT, which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria, Pseudomonas putida, P. aureofaciens, P. fluorescens and P. aeruginosa, tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure ‐ mean thickness, roughness, substratum coverage and surface to volume ratio ‐ showed that the four Pseudomonas strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies, which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum, and formed flat, uniform biofilms. P. aureofaciens resembled P. aeruginosa, but had a stronger tendency to form micro-colonies. Finally, the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0<03 mM, 0< 1m M or 0< 5m M citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover, biofilm roughness increased with lower citrate concentrations, whereas surface to volume ratio increased with higher citrate concentrations.

Bacterial respiration: a flexible process for a changing environment.

Abstract: The respiration of oxygen is fundamental to the life of higher animals and plants. The basic respiratory process in the mitochondria of these organisms involves the donation of electrons by low-redox-potential electron donors such as NADH. This is followed by electron transfer through a range of redox cofactors, bound to integral membrane or membrane-associated protein complexes. The process terminates in the reduction of the high-redox-potential electron acceptor, oxygen (Fig. 1). The free energy released during this electrontransfer process is used to drive the translocation of protons across the mitochondrial membrane to generate a trans-membrane proton electrochemical gradient or protonmotive force (∆p) that can drive the synthesis of ATP (Fig. 1). The respiratory flexibility of the mammalian mitochondrion is rather poor. There is some flexibility at the level of electron input (Fig. 1), but none at the level of electron output where cytochrome aa $ oxidase provides the only means of oxygen reduction. In the case of plant mitochondria, a slightly greater degree of respiratory flexibility is encountered with a number of alternative NADH dehydrogenases and two oxidases being apparent. This respiratory flexibility affords plant mitochondria with the capacity to contribute to processes other than the generation of ATP. For example, electron transfer from the alternative NADH dehydrogenase to the alternative oxidase is not coupled to the generation of ∆p and instead serves to release energy as heat, which can volatilize insect attractants to aid pollination. In the American skunk cabbage this same mechanism for heat production serves to permit growth at subzero temperatures (Nicholls & Ferguson, 1992). There is also some respiratory flexibility in the mitochondria of yeast, filamentous fungi and ancient protozoa, but it is amongst the Bacteria and Archaea that respiratory flexibility can be found at its most extreme. In these organisms, a diverse range of electron acceptors can be utilized including elemental sulphur and sulphur oxyanions (Hamilton, 1998), organic sulphoxides and sulphonates (Lie et al., 1999; McAlpine et al., 1998), nitrogen oxy-anions and nitrogen oxides (Berks et al., 1995), organic N-oxides (Czjzek et al., 1998), halogenated organics (Dolfing, 1990; Louie & Mohn, 1999; van de Pas et al., 1999), metalloid oxy-anions such as selenate and arsenate (Krafft & Macy, 1998; Macy et al., 1996, 1993; Schroder et al., 1997), transition metals such as Fe(III) and Mn(IV) (Lovley, 1991), and radionuclides such as U(VI) (Lovley & Phillips, 1992) and Tc(VII) (Lloyd et al., 1999). This respiratory diversity can be found amongst pyschrophiles, mesophiles and hyperthermophiles and contributes to the ability of prokaryotes to colonize many of Earth’s most hostile microoxic and anoxic environments.

Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement.

Abstract: Progress in the field of mycobacterial research has been hindered by the inability to readily generate defined mutant strains of the slow-growing mycobacteria to investigate the function of specific genes. An efficient method is described that has been used to generate several mutants, including the first double unmarked deletion strain of Mycobacterium tuberculosis. Four mutants were constructed: a marked deletion of the plcABC cluster, which encodes three phospholipases C; separate unmarked deletions in plcABC and tlyA (encoding a haemolysin); and a double unmarked mutant tlyAΔ plcABCΔ. To accomplish this, two series of vectors were designed, the first of which, named pNIL, allows manipulation of the target gene sequence at a variety of convenient restriction sites. The second series, named pGOAL, contains marker cassettes flanked by PacI restriction enzyme sites. The final suicide plasmid vectors were then obtained by cloning a marker cassette from a pGOAL vector into the single PacI site of the pNIL vector with the modified gene of interest. Finally, a two-step strategy was employed whereby single cross-over events were first selected, then screening for the second cross-over was carried out to yield the mutant strains. This technique will now allow the construction of potential vaccine strains without the inclusion of antibiotic resistance markers, the ability to make multiple defined mutations and the possibility of making more subtle defined mutations, such as point mutations.

Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes.

Abstract: Phylogenetic analysis of the genus Pseudomonas was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas (a total of 125 strains). Pseudomonas strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II). IGC I was further split into two subclusters, the ‘P. aeruginosa complex’, which included P. aeruginosa, P. alcaligenes, P. citronellolis, P. mendocina, P. oleovorans and P. pseudoalcaligenes, and the ‘P. stutzeri complex’, which included P. balearica and P. stutzeri. IGC II was further split into three subclusters that were designated the ‘P. putida complex’, the ‘P. syringae complex’ and the ‘P. fluorescens complex’. The ‘P. putida complex’ included P. putida and P. fulva. The ‘P. syringae complex’ was the cluster of phytopathogens including P. amygdali, P. caricapapayae, P. cichorii, P. ficuserectae, P. viridiflava and the pathovars of P. savastanoi and P. syringae. The ‘P. fluorescens complex’ was further divided into two subpopulations, the ‘P. fluorescens lineage’ and the ‘P. chlororaphis lineage’. The ‘P. fluorescens lineage’ contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, while the ‘P. chlororaphis lineage’ included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes formed a polyphyletic group within the ‘P. fluorescens complex’.

Autolysins of Bacillus subtilis: multiple enzymes with multiple functions.

Abstract: Autolysins are bacteriolytic enzymes that digest the cellwall peptidoglycan of the bacteria that produce them (Shockman&Ho$ ltje, 1994). Although potentially lethal, autolysins appear to be universal among bacteria that possess peptidoglycan. Peptidoglycan, the substrate of autolysins, is a polymer of amino sugars cross-linked by short peptides which forms a covalent matrix that surrounds the cytoplasmic membrane and constitutes the major skeletal component of the cell wall. It is critical in determining cell shape and preventing osmotic lysis under hypotonic conditions. Cell-wall peptidoglycan, whilst very strong, is also highly dynamic: the structure expands as the cell grows and is reshaped when it divides or differentiates. The possibility that autolysins are involved in selective removal of peptidoglycan has led to proposals that they are involved in numerous cellular processes including cell growth, cell-wall turnover, peptidoglycan maturation, cell division, separation, motility, chemotaxis, genetic competence, protein secretion, differentiation and pathogenicity (Foster, 1994; Blackman et al., 1998).

Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum

Abstract: Salmonella enterica is a facultative intracellular pathogen that is capable of causing disease in a range of hosts. Although human salmonellosis is frequently associated with consumption of contaminated poultry and eggs, and the serotypes Salmonella gallinarum and Salmonella pullorum are important world-wide pathogens of poultry, little is understood of the mechanisms of pathogenesis of Salmonella in the chicken. Type III secretion systems play a key role in host cell invasiveness and trigger the production of pro-inflammatory cytokines during invasion of mammalian hosts. This results in a polymorphonuclear cell influx that contributes to the resulting enteritis. In this study, a chicken primary cell culture model was used to investigate the cytokine responses to entry by the broad host range serotypes S. enteritidis and S. typhimurium, and the host specific serotype S. gallinarum, which rarely causes disease outside its main host, the chicken. The cytokines interleukin (IL)-1β, IL-2, IL-6 and interferon (IFN)-γ were measured by quantitative RT-PCR, and production of IL-6 and IFN-γ was also determined through bioassays. All serotypes were invasive and had little effect on the production of IFN-γ compared with non-infected cells; S. enteritidis invasion caused a slight down-regulation of IL-2 production. For IL-1β production, infection with S. typhimurium had little effect, whilst infection with S. gallinarum or S. enteritidis caused a reduction in IL-1β mRNA levels. Invasion of S. typhimurium and S. enteritidis caused an eight- to tenfold increase in production of the pro-inflammatory cytokine IL-6, whilst invasion by S. gallinarum caused no increase. These findings correlate with the pathogenesis of Salmonella in poultry. S. typhimurium and S. enteritidis invasion produces a strong inflammatory response, that may limit the spread of Salmonella largely to the gut, whilst S. gallinarum does not induce an inflammatory response and may not be limited by the immune system, leading to the severe systemic disease fowl typhoid.

Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa.

Abstract: A gene cluster similar to haem iron uptake loci of bacterial pathogens was identified in Pseudomonas aeruginosa. This phu locus (‘Pseudomonas haem uptake’) consisted of the phuR receptor gene and the phuSTUVW operon encoding a typical ABC transporter. Expression of phuR and phuSTUVW from mapped transcriptional-start sites occurred under iron-restricted growth conditions and was directly controlled by the Fur protein. Binding of Fur was demonstrated by DNase footprinting of two adjacent ‘Fur boxes’ that overlapped both the phuR and phuSTUVW promoters. Two tandem repeats of 154 bp were identified downstream of the phuSTUVW operon, each of which contained a strong Fur-dependent promoter driving expression of iron-regulated RNAs antisense to phuSTUVW. Mutant strains with deletions in phuR and phuSTUV showed greatly reduced growth with either haem or haemoglobin as the only iron source: the defects were complemented by plasmids harbouring the phuR or the phuSTUV genes, respectively. Deletions of phuW or of the tandem repeats had only minor effects on haem utilization. The remaining haem and haemoglobin uptake still observed in the ΔphuR or ΔphuSTUV deletion mutants was due to a second haem-acquisition system, has, which was also under the direct control of Fur. This second haem-receptor gene, hasR, was identified upstream of and in an operon with hasA, encoding a haem-binding extracellular protein. A ΔhasR mutant also exhibited decreased utilization of haem and haemoglobin, and a ΔphuR ΔhasR double mutant was virtually unable to take up either compound. Both the PhuR and HasR proteins were detected in the outer-membrane fraction of P. aeruginosa grown in low-iron media. Taken together, the evidence suggests that the phu and has loci encode two distinct systems required for the acquisition of haem and haemoglobin in P. aeruginosa.

The causes of Pseudomonas diversity

Abstract: The genus Pseudomonas encompasses arguably the most diverse and ecologically significant group of bacteria on the planet. Members of the genus are found in large numbers in all of the major natural environments (terrestrial, freshwater and marine) and also form intimate associations with plants and animals. This universal distribution suggests a remarkable degree of physiological and genetic adaptability.

Comparison of conserved structural and regulatory domains within divergent 16S rRNA-23S rRNA spacer sequences of cyanobacteria.

Abstract: PCR amplification of the internal transcribed spacer (ITS) between the 16S rRNA and 23S rRNA genes of the cyanobacterium Nostoc PCC 7120 gave three products. Two represented true ITS regions of different sizes, while the third was a heteroduplex. The longer spacer (ITS-L) contained 512 nucleotides and carried tRNAIle and tRNAAla genes, separated by a large stem–loop structure (V2) composed of short tandemly repeated repetitive sequences. Both tRNA genes, and the 5′ half of the intervening stem, were absent from the shorter spacer (ITS-S), of length 283 nucleotides, which was otherwise almost completely identical to ITS-L. The two spacer regions of Nostoc PCC 7120 were aligned to published ITS sequences of cyanobacteria, the cyanelle of Cyanophora paradoxa and Escherichia coli. Although the ITS regions of cyanobacteria vary in length from 283 to 545 nucleotides and contain either both tRNAIle and tRNAAla genes, only the tRNAIle gene, or neither, there is no correlation between ITS size and coding capacity for tRNAs. Putative secondary structures were determined for the deduced transcripts of the rrn operons of several cyanobacteria and were compared to that of E. coli. Highly conserved motifs important for folding and for maturation of the rRNA transcripts were identified, and regions homologous to bacterial antiterminators (box B–box A) were located. The conserved and variable regions of the cyanobacterial ITS are potential targets of PCR primers and oligonucleotide probes for detection and identification of cyanobacteria at different taxonomic levels.

Transcription factors in Candida albicans - environmental control of morphogenesis.

Abstract: Awareness of Candida albicans as a major human health threat has risen during recent years. Although infections by C. albicans can be relatively mild and superficial, systemic mycoses often occur in immunocompromised patients, or even as a consequence of long-term therapy with broad-spectrum antibiotics or of chemotherapy (reviewed by Odds, 1988). Effective antifungal agents which are free of side-effects are urgently needed. There is hope that recently developed techniques of manipulating C. albicans and the sequencing of its genome will lead to a thorough understanding of the virulence and biology of this fungal pathogen, thus offering the possibility of a knowledge-based approach to novel antifungal agents.

The amino acid/polyamine/organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations

Abstract: In this paper an analysis of 175 currently sequenced transport proteins that comprise the amino acid/polyamine/organocation (APC) superfamily is reported Members of this superfamily fall into 10 well-defined families that are either prokaryote specific, eukaryote specific or ubiquitous Most of these proteins exhibit 12 probable transmembrane spanners (TMSs), but members of two of these families deviate from this pattern, exhibiting 10 and 14 TMSs All members of these families are tabulated, their functional properties are reviewed and phylogenetic/sequence analyses define the evolutionary relationships of the proteins to each other Evidence is presented that the APC superfamily may include two other currently recognized families that exhibit greater degrees of sequence divergence from APC superfamily members than do the proteins of the 10 established families from each other At least some of the protein members of these two distantly related families exhibit 11 established TMSs Altogether, the APC superfamily probably includes 12 currently recognized families with members that exhibit exclusive specificity for amino acids and their derivatives but which can possess 10, 11, 12 or 14 TMSs per polypeptide chain

How Delisea pulchra furanones affect quorum sensing and swarming motility in Serratia liquefaciens MG1

Abstract: Halogenated furanones produced by the benthic marine macroalga Delisea pulchra inhibit swarming motility of Serratia liquefaciens MG1. This study demonstrates that exogenously added furanones control transcription of the quorum sensing regulated gene swrA in competition with the cognate signal molecule N-butanoyl-L-homoserine lactone. This in turn results in reduced production of the surface-active compound serrawettin W2, which is crucial for surface translocation of the differentiated swarm cells. It is demonstrated that furanones interfere with interspecies communication during swarming of mixed cultures and that the mode of interference in quorum-sensing control and interspecies communication is not through inhibition of autoinducer synthesis.

Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2-lacZ expression, glucanase resistance and thermotolerance.

Abstract: The protein kinase C (PKC1) pathway is essential for maintaining cell integrity in yeast. Here it is shown that various forms of cell wall damage result in activation of the downstream MAP kinase Slt2/Mpk1. Several cell wall mutants displayed enhanced FKS2-lacZ expression, a known output of Slt2 activation. A similar response was obtained with wild-type cells grown in the presence of the cell wall perturbants Calcofluor white and Zymolyase. Upregulation of FKS2-lacZ in response to sublethal concentrations of these agents fully depended on the presence of Slt2. The same cell wall stress conditions resulted in dual threonine and tyrosine phosphorylation of Slt2. Both Slt2 phosphorylation and FKS2-lacZ induction could be largely prevented by providing osmotic support to the plasma membrane. Interestingly, Slt2 phosphorylation in response to cell wall damage required the putative plasma-membrane-located sensor Mid2 but not Hcs77/Wsc1. Finally, cell wall perturbation gave rise to cells with increased resistance to glucanase digestion and heat shock. These responses depended on the presence of Slt2. These results indicate that weakening of the cell wall activates the Slt2/Mpk1 MAP kinase pathway and results in compensatory changes in the cell wall.

Experimental reproducibility in flow-chamber biofilms.

Abstract: The structural organization of microbial communities is influenced by many factors, e.g. nutrient composition, shear stress and temperature. This paper presents a general method for quantitative comparison of biofilm structures and assessment of experimental reproducibility between independent biofilm experiments. By using a novel computer program, COMSTAT, biofilm structures of Pseudomonas aeruginosa and an isogenic rpoS mutant were quantified. The strains were tagged with the green fluorescent protein (GFP) and grown in flow chambers with a defined minimal medium as substrate. Three independent rounds of biofilm experiments were performed and in each round, each of the two variants was grown in two separate channels. Nine image stacks were acquired in each channel 146 h after inoculation. An analysis of variance model incorporating the factors experiment round, bacterial strain, channel number and image stack number was used to analyse the data calculated by COMSTAT. Experimental reproducibility was verified by estimating the magnitude of the variance of the effects round (sigma(2)R) and the interaction between bacterial strain and round (sigma(2)BR). Mean thickness of the wild-type and rpoS mutant biofilms was estimated at 6.31 microm (SE 0.81 microm) and 16.85 microm (SE 0.87 microm), respectively.

Three pathways for trehalose biosynthesis in mycobacteria.

Abstract: Trehalose is present as a free disaccharide in the cytoplasm of mycobacteria and as a component of cell-wall glycolipids implicated in tissue damage associated with mycobacterial infection To obtain an overview of trehalose metabolism, we analysed data from the Mycobacterium tuberculosis genome project and identified ORFs with homology to genes encoding enzymes from three trehalose biosynthesis pathways previously characterized in other bacteria Functional assays using mycobacterial extracts and recombinant enzymes derived from these ORFs demonstrated that mycobacteria can produce trehalose from glucose 6-phosphate and UDP-glucose (the OtsA–OtsB pathway) from glycogen-like α(1→4)-linked glucose polymers (the TreY–TreZ pathway) and from maltose (the TreS pathway) Each of the pathways was found to be active in both rapid-growing Mycobacterium smegmatis and slow-growing Mycobacterium bovis BCG The presence of a disrupted treZ gene in Mycobacterium leprae suggests that this pathway is not functional in this organism The presence of multiple biosynthetic pathways indicates that trehalose plays an important role in mycobacterial physiology

The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects.

Abstract: Bacillus thuringiensis has been widely used for 40 years as a safe biopesticide for controlling agricultural pests and mosquitoes because it produces insecticidal crystal proteins. However, spores have also been shown to contribute to overall entomopathogenicity. Here, the opportunistic properties of acrystalliferous B. thuringiensis Cry− and Bacillus cereus strains were investigated in an insect species, Galleria mellonella, and in a mammal, BALB/c mice. In both animal models, the pathogenicity of the two bacterial species was similar. Mutant strains were constructed in which the plcR gene, encoding a pleiotropic regulator of extracellular factors, was disrupted. In larvae, co-ingestion of 106 spores of the parental strain with a sublethal concentration of Cry1C toxin caused 70% mortality whereas only 7% mortality was recorded if spores of the ΔplcR mutant strain were used. In mice, nasal instillation of 108 spores of the parental strain caused 100% mortality whereas instillation with the same number of ΔplcR strain spores caused much lower or no mortality. Similar effects were obtained if vegetative cells were used instead of spores. The cause of death is unknown and is unlikely to be due to actual growth of the bacteria in mice. The lesions caused by B. thuringiensis supernatant in infected mice suggested that haemolytic toxins were involved. The cytolytic properties of strains of B. thuringiensis and B. cereus, using sheep, horse and human erythrocytes and G. mellonella haemocytes, were therefore investigated. The level of cytolytic activity is highly reduced in ΔplcR strains. Together, the results indicate that the pathogenicity of B. thuringiensis strain 407 and B. cereus strain ATCC 14579 is controlled by PlcR.

Enzymically mediated bioprecipitation of uranium by a Citrobacter sp. : a concerted role for exocellular lipopolysaccharide and associated phosphatase in biomineral formation.

Abstract: A Citrobacter sp. accumulated uranyl ion (UO2(2+)) via precipitation with phosphate ligand liberated by phosphatase activity. The onset and rate of uranyl phosphate deposition were promoted by NH4(+), forming NH(4)UO(2)PO(4), which has a lower solubility product than NaUO(2)PO(4). This acceleration decoupled the rate-limiting chemical crystallization process from the biochemical phosphate ligand generation. This provided a novel approach to monitor the cell-surface-associated changes using atomic-force microscopy in conjunction with transmission electron microscopy and electron-probe X-ray microanalysis, to visualize deposition of uranyl phosphate at the cell surface. Analysis of extracted surface materials by (31)P NMR spectroscopy showed phosphorus resonances at chemical shifts of 0.3 and 2.0 p.p.m., consistent with monophosphate groups of the lipid A backbone of the lipopolysaccharide (LPS). Addition of fUO2(2+) to the extract gave a yellow precipitate which contained uranyl phosphate, while addition of Cd(2+) gave a chemical shift of both resonances to a single new resonance at 3 p.p.m. Acid-phosphatase-mediated crystal growth exocellularly was suggested by the presence of acid phosphatase, localized by immunogold labelling, on the outer membrane and on material exuded from the cells. Metal deposition is proposed to occur via an initial nucleation with phosphate groups localized within the LPS, shown by other workers to be produced exocellularly in association with phosphatase. The crystals are further consolidated with additional, enzymically generated phosphate in close juxtaposition, giving high loads of LPS-bound uranyl phosphate without loss of activity and distinguishing this from simple biosorption, or periplasmic or cellular metal accumulation mechanisms. Accumulation of 'tethered' metal phosphate within the LPS is suggested to prevent fouling of the cell surface by the accumulated precipitate and localization of phosphatase exocellularly is consistent with its possible functions in homeostatis and metal resistance.

InhA, a target of the antituberculous drug isoniazid, is involved in a mycobacterial fatty acid elongation system, FAS-II.

Abstract: Most drug-resistant clinical isolates of the tubercle bacillus are resistant to isoniazid, a first-line antituberculous drug. This antibiotic was shown to act on Mycobacterium tuberculosis by inhibiting a 2-trans-enoyl-acyl carrier protein reductase, called InhA. However, the exact role played by InhA in mycobacteria remained unclear. A mycobacterial enzyme fraction containing InhA was isolated. It displays a long-chain fatty acid elongation activity with the characteristic properties described for the FAS-II (fatty acid synthetase II) system. Inhibition of this activity by InhA inhibitors, namely isoniazid, hexadecynoyl-CoA or octadecynoyl-CoA, showed that InhA belongs to the FAS-II system. Moreover, the InhA inhibitors also blocked the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope. The data strongly suggest that isoniazid acts on the mycobacterial cell wall by preventing the FAS-II system from producing long-chain fatty acid precursors for mycolic acid biosynthesis.

The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase.

Abstract: Several different species of Pseudomonas: produce N:-acylhomoserine lactones (AHLs), quorum-sensing signal molecules which are involved in the cell-density-dependent control of secondary metabolite and virulence gene expression. When Pseudomonas fluorescens F113 was cross-streaked against AHL biosensors capable of sensitively detecting either short (C(4)-C(8)) or long (C(10)-C(14)) acyl chain AHLs, no activity was detectable. However, by extracting cell-free stationary-phase culture supernatants with dichloromethane followed by reverse-phase HPLC, three distinct fractions were obtained capable of activating the AHL biosensors. Three AHLs were subsequently characterized using high-resolution MS and chemical synthesis. These were (i) N:-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone (3OH, C(14:1)-HSL), a molecule previously known as the Rhizobium leguminosarum small bacteriocin as a consequence of its growth inhibitory properties, (ii) N:-decanoylhomoserine lactone (C(10)-HSL) and (iii) N:-hexanoylhomoserine lactone (C(6)-HSL). A gene (hdtS) capable of directing synthesis of all three P. fluorescens AHLs in Escherichia coli was cloned and sequenced. In vitro transcription/translation of hdtS yielded a protein of approximately 33 kDa capable of directing the synthesis of 3OH, C(14:1)-HSL, C(10)-HSL and C(6)-HSL in E. coli. HdtS does not belong to either of the known AHL synthase families (LuxI or LuxM) and is related to the lysophosphatidic acid acyltransferase family. HdtS may therefore constitute a member of a third protein family capable of AHL biosynthesis.

Spirochaetal lipoproteins and pathogenesis.

Abstract: Anchoring of proteins to membranes by lipid modification is a universal strategy of both prokaryotic and eukaryotic cells. The spirochaetes represent a distinct bacterial phylum that have utilized the lipoprotein design extensively as evidenced by genome-sequencing studies revealing a large number of different paralogous families of lipoprotein genes, encoding 22 potential lipoproteins in Treponema pallidum (Fraser et al., 1998) and 105 potential lipoproteins in Borrelia burgdorferi (Fraser et al., 1997). Roughly 8% of Bor. burgdorferi open reading frames are predicted to encode lipoproteins (Fraser et al., 1997), which is a significantly higher frequency than that of any other bacterial genome sequenced to date (The Institute for Genomic Research, 2000). An understanding of spirochaetal lipoproteins requires an appreciation of the distinctive doublemembrane architecture of spirochaetes, which shares characteristics of both Gram-positive and Gram-negative bacteria (see Fig. 1). As in Gram-positive bacteria, the cytoplasmic membrane of spirochaetes is closely associated with the peptidoglycan cell wall. Spirochaetes also have an outer membrane which provides a barrier shielding underlying antigens, such as the endoflagella, from the outside environment. However, the spirochaetal outer membrane appears to be fluid and labile, which contrasts it with the outer membrane of Gramnegative bacteria. This unique membrane architecture of spirochaetes and their ancient phylogeny suggest that the export, structure and function of spirochaetal lipoproteins have features which are unique to these organisms. As an indication of their importance in spirochaetes, lipoproteins are recognized to be the most abundant proteins in a wide array of organisms. Examples of lipoproteins that constitute a high percentage of the total protein composition of spirochaetes include OspA of Bor. burgdorferi, the Vmp proteins of the relapsing fever borreliae, SmpA of Brachyspira hyodysenteriae, LipL32 of the pathogenic Leptospira species and Tpp47 of T. pallidum. This paper is intended as a broad review of key issues involving spirochaetal lipoproteins, relating fundamental biochemical and physiological issues to those of pathogenesis. Rather than an exhaustive survey of current knowledge, the goal of this review is to highlight issues of current interest and point out areas for future research. Readers desiring more detailed information about spirochaetal lipoproteins are referred to additional sources, including websites such as the TIGR Microbial Database (The Institute for Genomic Research, 2000) and other reviews (e.g. Norris, 1993). The starting point for this review is lipoprotein secretion, processing and sorting, which will allow formulation of questions regarding how cellular destination is determined. With the exponential expansion of sequence data, what should be the criteria for identification of spirochaetal lipoproteins? Characterization of lipoproteins should also include determination of whether it is exported to the outer membrane and}or surface exposed, since surface-exposed lipoproteins are likely to be involved in how spirochaetes interact with their environment. Surface-exposed lipoproteins that have been implicated in essential roles in the interaction with the mammalian host will be reviewed. What are the environmental signals that regulate differential expression of lipoproteins? Some of the lipoproteins upregulated in the mammalian host undergo antigenic variation as a strategy for evading the host immune response. Lipoproteins are also involved in disease pathogenesis through their ability to trigger the host inflammatory response. Ultimately, lipoproteins are of interest as vaccines for prevention of spirochaetal infections.

Cryptosporidium parvum appears to lack a plastid genome.

Abstract: Surprisingly, unlike most Apicomplexa, Cryptosporidium parvum appears to lack a plastid genome. Primers based upon the highly conserved plastid small- or large-subunit rRNA (SSU/LSU rRNA) and the tufA-tRNAPhe genes of other members of the phylum Apicomplexa failed to amplify products from intracellular stages of C. parvum, whereas products were obtained from the plastid-containing apicomplexans Eimeria bovis and Toxoplasma gondii, as well as the plants Allium stellatum and Spinacia oleracea. Dot-blot hybridization of sporozoite genomic DNA (gDNA) supported these PCR results. A T. gondii plastid-specific set of probes containing SSU/LSU rRNA and tufA-tRNAPhe genes strongly hybridized to gDNA from a diverse group of plastid-containing organisms including three Apicomplexa, two plants, and Euglena gracilis, but not to those without this organelle including C. parvum, three kinetoplastids, the yeast Saccharomyces cerevisiae, mammals and the eubacterium Escherichia coli. Since the origin of the plastid in other apicomplexans is postulated to be the result of a secondary symbiogenesis of either a red or a green alga, the most parsimonious explanation for its absence in C. parvum is that it has been secondarily lost. If confirmed, this would indicate an alternative evolutionary fate for this organelle in one member of the Apicomplexa. It also suggests that unlike the situation with other diseases caused by members of the Apicomplexa, drug development against cryptosporidiosis targeting a plastid genome or metabolic pathways associated with it may not be useful.

Families of transmembrane transporters selective for amino acids and their derivatives.

Abstract: Twenty-one families of secondary [proton-motive force (PMF)-driven] carriers are herein described, which, in addition to 13 of the currently recognized 48 families within the ATP-dependent ATP-binding casette (ABC) superfamily and three families of channel proteins, largely account for the transport of amino acids and their derivatives (excluding proteins) into and out of all living cells on Earth. Family characteristics as well as structural and functional properties of the family constituents are described. By reference to our web site (http :}}www-biology.ucsd.edu}Cmsaier}transport}), sequences, phylogenetic relationships, detailed substrate-specificity information and the distribution of members of each family in 20 fully sequenced genomes of bacteria, archaea and eukaryotes are available. This review provides a comprehensive guide to the diversity of carriers that mediate the transport of amino acids and their derivatives across cell membranes.

Role of Pseudomonas aeruginosa PhoP-phoQ in resistance to antimicrobial cationic peptides and aminoglycosides.

Abstract: Resistance to the polycationic antibiotic polymyxin B and expression of the outer-membrane protein OprH in the opportunistic pathogen Pseudomonas aeruginosa both involve the PhoP-PhoQ two-component regulatory system. The genes for this system form an operon with oprH, oprH-phoP-phoQ, that responds to Mg2+ starvation and PhoP levels. In this study, the Mg2+-regulated promoter for this operon was mapped upstream of oprH by primer-extension experiments. An oprH::xylE-GmR mutant H855 was constructed and measurement of the catechol 2,3-dioxygenase activity expressed from this transcriptional fusion provided evidence for a second, weak promoter for phoP-phoQ. Wild-type P. aeruginosa PAO1 strain H103 was found to exhibit Mg2+-regulated resistance to the α-helical antimicrobial cationic peptide CP28 in addition to its previously characterized resistance to polymyxin B. Resistance to this peptide was unchanged in the OprH-null mutant H855 and a PhoP-null mutant H851. In contrast, PhoQ-null mutant H854 demonstrated constitutive CP28 resistance. Northern blot analysis revealed constitutive expression of phoP in this strain, implicating PhoP-PhoQ in the resistance of P. aeruginosa to cationic peptides. Furthermore, all three null-mutant strains demonstrated increased resistance to the aminoglycoside antibiotics streptomycin, kanamycin and amikacin. Two additional mutant strains, H895 and H896, were constructed that carried unmarked deletions in oprH and were found to exhibit aminoglycoside susceptibility equivalent to that of the wild-type. This result provided definitive evidence that OprH is not involved in P. aeruginosa aminoglycoside resistance and that the changes in resistance in strain H855 and a previously reported oprH mutant were due to polar effects on phoP-phoQ rather than loss of OprH expression. A role for PhoP-PhoQ in resistance to aminoglycosides is envisaged that is distinct from that in resistance to cationic peptides and polymyxin B.

A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole

Abstract: Azole resistance in Candida albicans can be mediated by several resistance mechanisms. Among these, alterations of the azole target enzyme and the overexpression of multidrug efflux transporter genes are the most frequent. To identify additional putative azole resistance genes in C. albicans, a genomic library from this organism was screened for complementation of fluconazole hypersusceptibility in Saccharomyces cerevisiae YKKB-13 lacking the ABC (ATP-binding cassette) transporter gene PDR5. Among the C. albicans genes obtained, a new gene was isolated and named FLU1 (fluconazole resistance). The deduced amino acid sequence of FLU1 showed similarity to CaMDR1 (formerly BEN(r)), a member of the major facilitator superfamily of multidrug efflux transporters. The expression of FLU1 in YKKB-13 mediated not only resistance to fluconazole but also to cycloheximide among the different drugs tested. The disruption of FLU1 in C. albicans had only a slight effect on fluconazole susceptibility; however, it resulted in hypersusceptibility to mycophenolic acid, thus suggesting that this compound could be a substrate for the protein encoded by FLU1. Disruption of FLU1 in a background of C. albicans mutants with deletions in several multidrug efflux transporter genes, including CDR1, CDR2 and CaMDR1, resulted in enhanced susceptibility to several azole derivatives. FLU1 expression did not vary significantly between several pairs of azole-susceptible and azole-resistant C. albicans clinical isolates. Therefore, FLU1 seems not to be required for the development of azole resistance in clinical isolates.

Cadmium uptake and subcellular compartmentation in the ectomycorrhizal fungus Paxillus involutus.

Abstract: Cadmium uptake and subcellular compartmentation in the ectomycorrhizal fungus Paxillus involutus were investigated using radiotracer flux analyses. Concentration-dependent Cd2+-uptake kinetics were characterized by a smooth, non-saturating curve that could be dissected into linear and saturable components. The linear-uptake kinetic component was interpreted as representing binding of Cd to apoplastic components, whereas the remaining saturable component was the result of carrier-mediated transport across the plasma membrane. Cell-wall-bound Cd was almost completely removed during desorption from cell-wall preparations. Cd2+ desorption from intact mycelium was found to be a function of time involving three compartments corresponding in series to cell wall (50%), cytoplasm (30%) and vacuole (20%), when mycelia were exposed to a 0.05 microM Cd concentration. At 4 degrees C, most of the Cd recovered was due to the cell-wall-bound fraction, suggesting that transport across the plasma membrane is a metabolically mediated process. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Cd accumulation in P. involutus mycelia by up to 28%, which indicates that transport of Cd2+ was partially dependent on the membrane potential. Cd2+ uptake into symplasm is linked to Ca2+ transport, as revealed by the inhibition of Cd accumulation by the Ca2+ ionophore A23187. The present work demonstrates the ability of the ectomycorrhizal fungus P. involutus to take up and further accumulate Cd in different compartments. Binding of Cd onto cell walls and accumulation of Cd in the vacuolar compartment may be regarded as two essential metal-detoxification mechanisms. These data represent a first step towards the understanding of metal-tolerance mechanisms in mycorrhizal fungi.

Proteome analysis of Bacillus subtilis extracellular proteins: a two-dimensional protein electrophoretic study.

Abstract: To analyse the proteome of Bacillus subtilis extracellular proteins, extracellular protein samples were prepared from culture media (minimal medium containing 0·4% glucose) of parental B. subtilis 168, a secA-temperature sensitive mutant and an ffh conditional mutant, and examined by two-dimensional gel electrophoresis. Approximately 100 to 110 spots were visualized in a gel of B. subtilis 168 extracellular proteins. Over 90% and 80% of these disappeared in the absence of SecA and Ffh, respectively. Thirty-eight obvious spots on the gel of the B. subtilis 168 preparation were selected and compared with spots obtained under SecA- or Ffh-deficient conditions. The appearance of 36 of these 38 spots depended on SecA and Ffh. Nineteen additional extracellular proteins were detected in cultures maintained in cellobiose, maltose and soluble starch. Among 23 proteins of which the N-terminal amino acid sequences were determined, 17 were extracellular proteins having signal peptides in their precursor form. Two membrane proteins, YfnI and YflE, were cleaved behind 226Ala-Tyr-Ala228 and 213Ala-Leu-Ala215, respectively, and of which products seemed to be liberated into the culture medium. The production of YfnI and YflE were also dependent on SecA and Ffh. These results indicate that most extracellular proteins target to and translocate across the cytoplasmic membrane by co-operation between the signal-recognition particle and Sec protein-secretion pathways. In contrast, a spot for Hag appeared independent from SecA and Ffh. Intracellular proteins Gap, SodA and KatA were identified in the extracellular protein samples. On the basis of these results and computer searches, it was predicted that B. subtilis produces 150 to 180 proteins extracellularly.

Use of green fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in vivo and in vitro fish models

Abstract: Edwardsiella tarda is a fish pathogen that causes systemic infections in many food and ornamental fish. E. tarda PPD130/91 and PPD125/87 were selected as representatives of the virulent and avirulent groups, respectively, from eight fish isolates, and transformed with plasmids encoding either green fluorescent protein (pGFPuv) or blue fluorescent protein (pBFP2). Two host models were used to study the invasion pathway of E. tarda in vitro and in vivo. Epithelioma papillosum of carp (EPC) was used as the first model. Virulent and avirulent E. tarda strains were found to adhere to and invade EPC cells. Interactions between E. tarda and host cells examined under confocal microscopy and intracellular growth were followed at different time points. Bacterial internalization of PPD130/91 and PPD125/87 involved microfilaments and protein tyrosine kinase since cytochalasin D (an inhibitor of microfilament polymerization) and genistein (an inhibitor of protein tyrosine kinase) prevented internalization. Confocal studies revealed co-localization of polymerized actin with bacteria. Staurosporine, a protein kinase C inhibitor, accelerated internalization of PPD125/87, whereas PD098059, a mitogen-activated protein kinase (MAPK) kinase inhibitor prevented internalization of PPD130/91. In the second model, blue gourami were infected with E. tarda intramuscularly. Mortalities were observed in PPD130/91(pGFPuv)-infected fish with high bacterial numbers detectable in all organs. PPD125/87(pBFP2)-infected fish did not die and the bacterial population decreased over time. Mixed infections comprised of both PPD130/91(pGFPuv) and PPD125/87(pBFP2), where inoculum size was similar to the single infections, caused mortalities in fish. High bacterial populations were noted only in the fish body muscle. The PPD125/87(pBFP2) population in the fish decreased after 5 d. The number of PPD130/91(pGFPuv) also decreased in the fish organs, except for continued high growth in the body muscle. Histology revealed necrosis of the tissue (body muscle and liver) and fluorescent bacteria in fish that were infected with PPD130/91(pGFPuv) but not with PPD125/87(pBFP2). This study showed that fluorescent proteins are a useful tool for investigating bacterial host cell infection, and information elucidated here sheds new light on the interactions between E. tarda and its hosts.

Development of oligonucleotide probes and PCR primers for detecting phylogenetic subgroups of sulfate-reducing bacteria

Abstract: PCR primer sets for the 16S rRNA gene of six phylogenetic groups of sulfate-reducing bacteria (SRB) were designed. Their application in conjunction with group-specific internal oligonucleotide probes was used to detect SRB DNA in samples of landfill leachate. Six generic/suprageneric groups could be differentiated: Desulfotomaculum; Desulfobulbus; Desulfobacterium; Desulfobacter; Desulfococcus–Desulfonema–Desulfosarcina; Desulfovibrio–Desulfomicrobium. The predicted specificities of the PCR primer and oligonucleotide probe combinations were confirmed with DNA from reference strains. In all cases, the PCR primers and probes were specific, the only exception being that the Desulfococcus–Desulfonema–Desulfosarcina (group 5) PCR primers were able to amplify DNA from Desulfobacterium (group 3) reference strains but these groups could nevertheless be differentiated with the internal oligonucleotide probes. The proliferation of SRB in landfill sites interferes with methanogenesis and waste stabilization, but relatively little is known about the composition of SRB populations in this environment. DNA was extracted from samples of landfill leachate from several municipal waste landfill sites and used as template in PCR reactions with SRB group-specific primer sets. Group-specific oligonucleotide probes were then used to confirm that the PCR products obtained contained the target SRB 16S rDNA. Both ‘direct’ and ‘nested’ PCR protocols were used to amplify SRB 16S rDNA from landfill leachates. Three of the six SRB groups could be detected using the ‘direct’ PCR approach (Desulfotomaculum, Desulfobacter and Desulfococcus–Desulfonema–Desulfosarcina). When ‘nested’ PCR was applied, an additional two groups could be detected (Desulfobulbus and Desulfovibrio–Desulfomicrobium). Only Desulfobacterium could not be detected in any leachate samples using either direct or nested PCR. The SRB-specific 16S rDNA primers and probes described here can be applied to investigations of SRB molecular ecology in general, and can be further developed for examining SRB population composition in relation to landfill site performance.

Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography-tandem mass spectrometry.

Abstract: Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates. To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography–tandem mass spectrometry (μLC/MS/MS). The isolates selected for this analysis were from the sputum of a CF patient: strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections. Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis. Conditions of protein extraction were optimized for consistent high-resolution separation of several hundred proteins from these clinical isolates as detected by Coomassie staining of 2-D gels. Fourteen proteins were selected for analysis; this group included those whose expression was common between both strains as well as unique for each strain. The proteins were identified by μLC/MS/MS of the peptides produced by an in-gel tryptic digestion and compared to translated data from the Pseudomonas Genome Project; optimization of this technique has allowed for the comparison of proteins expressed by strains 383 and 2192.