Combined physical and genetic map of the Pseudomonas putida KT2440 chromosome

A combined physical and genetic map of the Pseudomonas putida KT2440 genome was constructed from data obtained by pulsed-field gel electrophoresis techniques (PFGE) and Southern hybridization. Circular genome size was estimated at 6.0 Mb by adding the sizes of 19 SwaI, 9 PmeI, 6 PacI, and 6 I-CeuI fragments. A complete physical map was achieved by combining the results of (i) analysis of PFGE of the DNA fragments resulting from digestion of the whole genome with PmeI, SwaI, I-CeuI, and PacI as well as double digestion with combinations of these enzymes and (ii) Southern hybridization analysis of the whole wild-type genome digested with different enzymes and hybridized against a series of probes obtained as cloned genes from different pseudomonads of rRNA group I and Escherichia coli, as P. putida DNA obtained by PCR amplification based on sequences deposited at the GenBank database, and by labeling of macrorestriction fragments of the P. putida genome eluted from agarose gels. As an alternative, 10 random mini-Tn5-Km mutants of P. putida KT2440 were used as a source of DNA, and the band carrying the mini-Tn5 in each mutant was identified after PFGE of a series of complete chromosomal digestions and hybridization with the kanamycin resistance gene of the mini-Tn5 as a probe. We established a circular genome map with an average resolution of 160 kb. Among the 63 genes located on the genetic map were key markers such as oriC, 6 rrn loci (rnnA to -F), recA, ftsZ, rpoS, rpoD, rpoN, and gyrB; auxotrophic markers; and catabolic genes for the metabolism of aromatic compounds. The genetic map of P. putida KT2440 was compared to those of Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens SBW25. The chromosomal backbone revealed some similarity in gene clustering among the three pseudomonads but differences in physical organization, probably as a result of intraspecific rearrangements.     

Bacterial whole cell protein profiles of the rRNA group II pseudomonads

Studies on bacterial whole cell protein profiles showed that members of the rRNA group II pseudomonads were distinct from other non-fluorescent and fluorescent pseudomonads, including Pseudomonas aeruginosa, the type species of the genus Pseudomonas. Strains of Ps. andropogonis, Ps. caryophylli, Ps. gladioli pv. gladioli, Ps. pickettii, Ps. pseudomallei and Ps. rubrisubalbicans showed uniform and distinct protein patterns, while strains of Ps. solanacearum and Ps. cepacia displayed differences within species. Numerical analysis of their protein profiles with GelManager and Taxan programs generated dendrograms comprising 16 clusters at 89% similarity. Each cluster included strains belonging to the same species with the exception of Ps. solanacearum, which fragmented into three clusters. Pseudomonas solanacearum showed different protein patterns correlating with different biovars and the two divisions of Cook et al. (1989), as well as the results of 16S rRNA gene sequencing. The whole cell protein profiles of a total of 83 strains belonging to 14 bacterial species were numerically analysed.     

The diversity of lipases from psychrotrophic strains of Pseudomonas: a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens

Strains of Pseudomonas fluorescens and Ps. fragi are the predominant psychrotrophs found in raw milk and may cause spoilage due to the secretion of hydrolytic enzymes such as lipase and protease. The diversity of lipases has been examined in Pseudomonas isolates from raw milk which represent different taxonomic groups (phenons). Significant diversity was found using both DNA hybridization and immunoblotting techniques, which has implications for the development of a diagnostic test. The lipase-encoding gene (lipA) was cloned from one strain, C9, of Ps. fluorescens biovar V. In contrast to previously reported lipase sequences from Ps. fluorescens, the gene encodes a lipase of M(r) 33 kDa. Alignment of all known Pseudomonas and Burkholderia lipase amino acid sequences indicates the existence of two major groups, one of M(r) approximately 30 kDa comprising sequences from Ps. fragi, Ps. aeruginosa, Ps. fluorescens C9 and Burkholderia, and one of approximately 50 kDa comprising Ps. fluorescens lipases. The lipase from C9 does not contain a signal peptide and is presumed to be secreted via a signal peptide-independent pathway. The lipA gene of strain C9 was disrupted by insertional mutagenesis. The mutant retained its lipolytic phenotype, strongly suggesting the presence of a second lipase in this strain.     

Surveillance of sexual behaviour among homosexual men in a central London health authority

Flagellin gene central regions from 111 isolates of Pseudomonas fluorescens SBW25 obtained from soil during a field release experiment were analysed using a combined PCR/RFLP technique to look for variation. In addition, a 858 bp flagellin gene sequence from the original strain and the last isolate obtained from the release site were compared. There was no variation in flagellin gene sequences indicating that the gene was stable over the period of the release, and that the flagellin gene is a suitable marker for use in the detection of bacteria in release experiments. A comparison of Ps. fluorescens SBW25 flagellin with other sequenced flagellins revealed closest homology to the flagellin of Ps. putida PRS2000.     

Down regulation of yeast G protein-coupled receptors

In this research, totally 200 raw milk samples in different areas of Ankara were collected from various dairy plant. The isolated psychotrophic bacteria from the raw milk samples are the species of Pseudomonas spp., Acinetobacter spp., Alcaligenes and Aeromonas. Isolation of Pseudomonas and other gram(-) psychrotrophic bacteria types are determined as P. aeruginosa 11 (5.5%), P. putida 11 (5.5%), P. fluorescens biotype I 10 (5.0%), P. fluorescens biotype II 4 (2.0%), P. fluorescens biotype III 6 (3.0%), P. aurefaciens 2 (1.0%), P. pseudomallei 3 (1.5%), P. cepacia 1 (0.5%), A. calcoaceticus lowffii 5 (2.5%), A. calcoaceticus anitratum 4 (2.0%), A. faecalis 3 (1.5%) and A. hydrophilia 1 (0.5%).     

Missouri Advantage

The OprB porin-mediated glucose transport system was investigated in Pseudomonas chlororaphis, Burkholderia cepacia, and Pseudomonas fluorescens. Kinetic studies of [U-14C]glucose uptake revealed an inducible system of low Km values (0.3-5 microM) and high specificity for glucose. OprB homologs were purified and reconstituted into proteoliposomes. The porin function and channel preference for glucose were demonstrated by liposome swelling assays. Examination of the periplasmic glucose-binding protein (GBP) components by Western immunoblotting using P. aeruginosa GBP-specific antiserum revealed some homology between P. aeruginosa GBP and periplasmic proteins from P. fluorescens and P. chlororaphis but not B. cepacia. Circular dichroism spectropolarimetry of purified OprB-like porins from the three species revealed beta sheet contents of 31-50% in agreement with 40% beta sheet content for the P. aeruginosa OprB porin. These findings suggest that the high-affinity glucose transport system is primarily specific for glucose and well conserved in the genus Pseudomonas although its outer membrane component may differ in channel architecture and specificity for other carbohydrates.     

Sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6, and V9 domains reveal highly species-specific variations within the genus Agrocybe

A comparative study of variable domains V4, V6, and V9 of the mitochondrial small-subunit (SSU) rRNA was carried out with the genus Agrocybe by PCR amplification of 42 wild isolates belonging to 10 species, Agrocybe aegerita, Agrocybe dura, Agrocybe chaxingu, Agrocybe erebia, Agrocybe firma, Agrocybe praecox, Agrocybe paludosa, Agrocybe pediades, Agrocybe alnetorum, and Agrocybe vervacti. Sequencing of the PCR products showed that the three domains in the isolates belonging to the same species were the same length and had the same sequence, while variations were found among the 10 species. Alignment of the sequences showed that nucleotide motifs encountered in the smallest sequence of each variable domain were also found in the largest sequence, indicating that the sequences evolved by insertion-deletion events. Determination of the secondary structure of each domain revealed that the insertion-deletion events commonly occurred in regions not directly involved in the secondary structure (i.e., the loops). Moreover, conserved sequences ranging from 4 to 25 nucleotides long were found at the beginning and end of each domain and could constitute genus-specific sequences. Comparisons of the V4, V6, and V9 secondary structures resulted in identification of the following four groups: (i) group I, which was characterized by the presence of additional P23-1 and P23-3 helices in the V4 domain and the lack of the P49-1 helix in V9 and included A. aegerita, A. chaxingu, and A. erebia; (ii) group II, which had the P23-3 helix in V4 and the P49-1 helix in V9 and included A. pediades; (iii) group III, which did not have additional helices in V4, had the P49-1 helix in V9 and included A. paludosa, A. firma, A. alnetorum, and A. praecox; and (iv) group IV, which lacked both the V4 additional helices and the P49-1 helix in V9 and included A. vervacti and A. dura. This grouping of species was supported by the structure of a consensus tree based on the variable domain sequences. The conservation of the sequences of the V4, V6, and V9 domains of the mitochondrial SSU rRNA within species and the high degree of interspecific variation found in the Agrocybe species studied open the way for these sequences to be used as specific molecular markers of the Basidiomycota.     

Biodiversity of Pneumocystis carinii hominis: typing with different DNA regions

The purpose of this study was to identify the most useful gene for the detection of biodiversity of Pneumocystis carinii hominis isolates and to compare samples from French and Italian subjects. We studied 20 bronchoalveolar lavage fluid specimens from 20 human immunodeficiency virus-infected patients (10 French and 10 Italian patients) with Pneumocystis carinii pneumonia by DNA sequencing of the thymidylate synthase (TS), 5S rRNA, large-subunit mitochondrial rRNA (mt LSU rRNA), and internal transcribed spacer (ITS1 and ITS2) genes. Thirteen of the 20 sequenced samples had the prototype TS gene sequence. Fourteen of the 20 samples showed the prototype sequence of the 5S rRNA gene, and 6 had variant sequences of the 5S rRNA gene. The mt LSU rRNA gene was sequenced for 18 of the 20 samples; all sequences were different from the prototype sequence and were classified into four groups. Thirteen of the 20 ITS1 and ITS2 sequences were analyzed, and all the sequences were found to be different from the prototype sequence and were classified into 10 groups. The internal transcribed spacer regions thus appear to be the most discriminatory region of DNA for analysis of the biodiversity of P. carinii hominis isolates.     

In vitro antibacterial activity and susceptibility of cefsulodin, an antipseudomonal cephalosporin, to beta-lactamases

Cefsulodin sodium (SCE-129, CGP-7174/E), active in minimum inhibitory concentrations (MICs) of 0.5 to 64 microgram/ml, was about 16- to 32-fold more active than carbenicillin against Psuedomonas aeruginosa. It was also active against P. diminuta, P. maltophilia, P. paucimobilis, and P. pseudoalcaligenes (MICs of 1 to 32 microgram/ml) but not against other species of Pseudomonas or other gram-negative bacteria. Except with highly carbenicillin-resistant isolates, MICs of cefsulodin for P. aeruginosa were little affected by an increase in the inoculum. With a small inoculum, minimum bactericidal concentrations (MBCs) were the same as or twice the MIC, but increasing the inoculum had a greater effect on the MBC than on the MIC. Cefsulodin was not hydrolyzed by the beta-lactamase induced in P. aeruginosa by growth in the presence of benzylpenicillin and was a poor substrate for beta-lactamases from Enterobacter cloacae and Proteus morganii. However, it was hydrolyzed, albeit slowly, by the beta-lactamase produced by most of our highly carbenicillin-resistant isolates of P. aeruginosa and by TEM-type beta-lactamases.     

Phylogenetic relationships and diversity within the Pasteurella haemolytica complex based on 16S rRNA sequence comparison and outer membrane protein and lipopolysaccharide analysis

The outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles of 30 untypeable isolates of Pasteurella haemolytica were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with the profiles of typeable isolates. The phylogenetic relationships of 28 isolates representing each of the serotypes of P. haemolytica and Pasteurella trehalosi, as well as untypeable isolates of P. haemolytica, were determined by comparing 16S rRNA sequences. The analysis of the OMP and LPS profiles of the untypeable isolates revealed five groups, which were designated untypeable groups 1 (UG1) through UG5. The UG1 and UG2 isolates had OMP and LPS profiles identical to the profiles of certain serotype A1 and A2 isolates, respectively. Furthermore, UG1 isolates originating from cattle and sheep could be clearly differentiated on the basis of their OMP profiles. The OMP and LPS profiles of UG3 isolates were similar appearance to the profiles of serotype A11 isolates, suggesting that these two groups are closely related. The OMP profiles of UG4 and UG5 isolates were unique and different from the OMP profiles of the UG1 through UG3 isolates. A comparison of 16S rRNA sequences revealed that typeable isolates of P. haemolytica could be divided into the following three groups: (i) serotype A1, A5 through A9, A12 through A14, and A16 isolates, (ii) serotype A2 isolates, and (iii) serotype A11 isolates. the isolates belonging to the first group all had identical sequences, whereas the sequences of isolates belonging to the second and third groups differed from the sequences of the isolates belonging to the first group at two and four base positions, respectively. The sequence data for the untypeable isolates confirmed the conclusions derived from the OMP and LPS analysis. Isolates belonging to UG1 and UG2 were identical to serotype A1 and A2 isolates, respectively; isolates belonging to UG3 were related to serotype A11 isolates, although there was some sequence heterogeneity within this group; and isolates belonging to UG4 and UG5 were more distantly related to P. haemolytica than were isolates belonging to UG1 through UG3 and were clearly members of two different species. As expected, isolates of P. trehalosi were event more distantly related to P. haemolytica than were the untypeable isolates, but there was significantly more sequence variation among the four serotypes of this species than there was among the serotypes of P. haemolytica. The correlation of the OMP and LPS data with the 16S rRNA sequence data suggested that OMP and LPS analyses might be useful for preliminary screening and comparing large numbers of isolates in taxonomic and epidemiological studies of the Pasteurellaceae.     

Evaluation and characterization of micronuclei in early spermatids of mice exposed to 1,3-butadiene

The 18S rRNA gene (Rns) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1-T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5-T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.     

Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria

Many gram-negative bacteria regulate expression of specialized gene sets in response to population density. This regulatory mechanism, called autoinduction or quorum-sensing, is based on the production by the bacteria of a small, diffusible signal molecule called the autoinducer. In the most well-studied systems the autoinducers are N-acylated derivatives of L-homoserine lactone (acyl-HSL). Signal specificity is conferred by the length, and the nature of the substitution at C-3, of the acyl side-chain. We evaluated four acyl-HSL bioreporters, based on tra of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudomonas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HSLs, and alkanoyl-HSLs with chain lengths ranging from C4 to C12. The traG::lacZ fusion reporter from the A. tumefaciens Ti plasmid was the single most sensitive and versatile detector of the four. Using this reporter, we screened 106 isolates representing seven genera of bacteria that associate with plants. Most of the Agrobacterium, Rhizobium, and Pantoea isolates and about half of the Erwinia and Pseudomonas isolates gave positive reactions. Only a few isolates of Xanthomonas produced a detectable signal. We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography. Among the pseudomonads and erwinias, most produced a single dominant activity chromatographing with the properties of N-(3-oxo-hexanoyl)-L-HSL. However, a few of the erwinias, and the P. fluorescens and Ralstonia solanacearum isolates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlike any of our standards. The few positive xanthomonas, and almost all of the agrobacteria, produced small amounts of a compound with the chromatographic properties of N-(3-oxo-octanoyl)-L-HSL. Members of the genus Rhizobium showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals. Several isolates produced extremely nonpolar compounds indicative of very long acyl side-chains. Production of these compounds suggests that quorum-sensing is common as a gene regulatory mechanism among gram-negative plant-associated bacteria.     

The phylogeny of unicellular, extremely halotolerant cyanobacteria

We examined the morphology, physiology, and 16S rRNA gene sequences of three culture collection strains and of ten novel isolates of unicellular cyanobacteria from hypersaline environments. The strains were morphologically diverse, with average cell widths ranging from 2.8 to 10.3 micron. There were single-celled, colonial, and baeocyte-forming strains. However, morphological traits were markedly variable with culture conditions. In contrast, all strains displayed extreme halotolerance (growing close to optimally at above 12% salinity); all were obligately marine, euryhaline, and moderately thermophilic; and all shared a suite of chemotaxonomic markers including phycobilins, carotenoids, and mycosporine-like amino acids. 16S rRNA gene sequence analysis indicated that the strains were related to each other. Sequence similarity analysis placed the strains in a monophyletic cluster (which we named the Halothece cluster) apart from all cultured or uncultured, not extremely halotolerant cyanobacteria whose 16S rRNA gene sequences are available in public nucleotide sequence databases. This represents the first case in which a phylogenetically coherent group of cyanobacteria can be defined on the basis of physiology. The Halothece cluster contained two subclusters that may be divergent at the generic level, one encompassing 12 strains (spanning 5% 16S rRNA gene sequence divergence and named the Euhalothece subcluster), and a single deep-branching isolate. Phenotypic characterization of the isolates, including morphological, physiological, and chemotaxonomic traits, did not distinguish these subclusters and only weakly suggested the existence of two separate clades, one encompassing strains of small cell size (cell width < 5 m) and another one encompassing strains of larger cell size.     

Transformation of elemental mercury by bacteria

The fate and impact of elemental mercury in closed bacterial cultures were examined. The quantity of elemental mercury oxidized by bacteria ranged from small amounts for Pseudomonas aeruginosa, P. fluorescens, Escherichia coli, and Citrobacter to essentially all of the added elemental mercury for Bacillus subtilis and B. megaterium. The percentage of the total mercury in the system associated with bacterial cells ranged from 18.6 to 43.2%. Growth of the two Pseudomonas species was inhibited by elemental mercury, whereas growth of the other cultures was not distinguishable from that in mercury-free controls. No methylmercury was formed by the six cultures within 48 h.     

Acquisition of a deliberately introduced phenol degradation operon, pheBA, by different indigenous Pseudomonas species

Horizontal transfer of genes of selective value in an environment 6 years after their introduction into a watershed has been observed. Expression of the gene pheA, which encodes phenol monooxygenase and is linked to the pheBA operon (A. Nurk, L. Kasak, and M. Kivisaar, Gene 102:13-18, 1991), allows pseudomonads to use phenol as a growth substrate. Pseudomonas putida strains carrying this operon on a plasmid were used for bioremediation after an accidental fire in the Estonia oil shale mine in Estonia in 1988. The water samples used for studying the fate of the genes introduced were collected in 1994. The same gene cluster was also detected in Pseudomonas strains isolated from water samples of a nearby watershed which has been continuously polluted with phenols due to oil shale industry leachate. Together with the more frequently existing counterparts of the dmp genes (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), the pheA gene was also represented in the phenol-degrading strains. The area where the strains containing the pheA gene were found was restricted to the regular route of phenolic leachate to the Baltic Sea. Nine Pseudomonas strains belonging to four different species (P. corrugata, P. fragi, P. stutzeri, and P. fluorescens biotypes B, C, and F) and harboring horizontally transferred pheBA operons were investigated. The phe genes were clustered in the same manner in these nine phe operons and were connected to the same promoter as in the case of the original pheBA operon. One 10.6-kb plasmid carrying a pheBA gene cluster was sequenced, and the structure of the rearranged pheBA operon was described. This data indicates that introduced genetic material could, if it encodes a beneficial capability, enrich the natural genetic variety for biodegradation.     

Sequence variation in the small-subunit rRNA gene of Plasmodium malariae and prevalence of isolates with the variant sequence in Sichuan, China

By two PCR-based diagnostic methods, Plasmodium malariae infections have been rediscovered at two foci in the Sichuan province of China, a region where no cases of P. malariae have been officially reported for the last 2 decades. In addition, a variant form of P. malariae which has a deletion of 19 bp and seven substitutions of base pairs in the target sequence of the small-subunit (SSU) rRNA gene was detected with high frequency. Alignment analysis of Plasmodium sp. SSU rRNA gene sequences revealed that the 5' region of the variant sequence is identical to that of P. vivax or P. knowlesi and its 3' region is identical to that of P. malariae. The same sequence variations were also found in P. malariae isolates collected along the Thai-Myanmar border, suggesting a wide distribution of this variant form from southern China to Southeast Asia.     

Effect of cultivation medium on some physicochemical parameters of outer bacterial membrane

The changes of surface charge and hydrophobicity of the outer bacterial membrane in relation to utilization of n-hexadecane were studied. For this spectrophotometric study adsorption of methylene blue and transport of gentian violet were used. The decrease in the negative charge of the bacterial strains Pseudomonas putida CCM 3423, P. aeruginosa, and P. fluorescens CCM 2115, depended on the type of growth medium. The decrease of surface charge was in the order: meat extract peptone broth > mineral medium with glucose > mineral medium with n-hexadecane. The highest permeability of the bacterial membrane for gentian violet was determined in the case of P. fluorescens grown in meat extract peptone broth. This effect can be explained by a greater hydrophobicity of the bacterial surface for this strain. In other strains a lower permeability was observed. P. fluorescens showed a greater adherence to hexadecane.     

Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon

Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae. The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented. A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria. Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group. Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species. A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology.