Sequence and relatedness in other bacteria of the Pseudomonas aeruginosa oprP gene coding for the phosphate-specific porin P

The oprP gene encoding the Pseudomonas aeruginosa phosphate-specific outer membrane porin protein OprP was sequenced. Comparison of the derived amino acid sequence with the known sequences of other bacterial porins demonstrated that OprP could be no better aligned to these porin sequences than it could to the periplasmic phosphate-binding protein PhoS of Escherichia coli. Southern hybridization and restriction mapping of the oprP gene in 37 clinical isolates and the 17 serotype strains of P. aeruginosa revealed that restriction sites in the vicinity of the oprP gene were highly conserved. Several species from the Pseudomonas fluorescens rRNA homology group contained DNA that hybridized to an oprP gene probe.     

Use of a chiA probe for detection of chitinase genes in bacteria from the Chesapeake Bay(1)

PCR primers specific for the chiA gene were designed by alignment and selection of highly conserved regions of chiA sequences from Serratia marcescens, Alteromonas sp., Bacillus circulans and Aeromonas caviae. These primers were used to amplify a 225 bp fragment of the chiA gene from Vibrio harveyi to produce a chiA gene probe. The chiA PCR primers and probe were used to detect the presence of the chiA gene in an assemblage of 53 reference strains and gave consistent results. Selected chiA fragments amplified by PCR were cloned and sequenced from nine known strains and from Chesapeake Bay isolates 6d and 11d. This confirmed the specificity and utility of the primers for detection of chiA-positive environmental strains. Over 1000 bacterial isolates from Chesapeake Bay water samples were tested for the presence of the chiA gene which was found to be present in 5-41% (average 21%) of the culturable bacterial community. The approach developed in this study was valuable for isolation and enumeration of chiA-positive bacteria in environmental samples.     

Specific detection of Pseudomonas spp. in milk by fluorescence in situ hybridization using ribosomal RNA directed probes

AIMS: Pseudomonas spp. are considered the most important milk spoilage organisms. Here we describe development of a fluorescence in situ hybridization (FISH) probe specific for detection and enumeration of Pseudomonas spp. in milk. METHODS AND RESULTS: 16S rRNA sequences were analysed to develop specific oligonucleotide probe for the genus Pseudomonas. Twenty different Pseudomonas spp. and 23 bacterial species from genera other than Pseudomonas (as negative controls) were tested. All tested Pseudomonas spp. yielded a positive FISH reaction, whereas negative controls showed no FISH reaction except for Burkholderia cepacia that showed a relatively weak FISH reaction. The FISH assay specifically stains Pseudomonas in milk when the milk contains a mixture of other bacterial species. The FISH assay takes 2 h and compares favourably with current culturing methods, which take a minimum of 48 h. Specificity of the probe was validated using polymerase chain reaction to selectively amplifying the Pseudomonas rDNA gene and sequencing the gene products. CONCLUSIONS: The method presented in this study allows simultaneously detection, identification and enumeration of Pseudomonas spp. in milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and accurate enumeration of Pseudomonas facilitates the identification of specific contamination sources in dairy plants, the accurate validation of pasteurization treatments and the prediction of shelf life of processed milk.     

Flagellin gene sequence variation in the genus Pseudomonas

Flagellin gene (fliC) sequences from 18 strains of Pseudomonas sensu stricto representing 8 different species, and 9 representative fliC sequences from other members of the gamma sub-division of proteobacteria, were compared. Analysis was performed on N-terminal, C-terminal and whole fliC sequences. The fliC analyses confirmed the inferred relationship between P. mendocina, P. oleovorans and P. aeruginosa based on 16S rRNA sequence comparisons. In addition, the analyses indicated that P. putida PRS2000 was closely related to P. fluorescens SBW25 and P. fluorescens NCIMB 9046T, but suggested that P. putida PaW8 and P. putida PRS2000 were more closely related to other Pseudomonas spp. than they were to each other. There were a number of inconsistencies in inferred evolutionary relationships between strains, depending on the analysis performed. In particular, whole flagellin gene comparisons often differed from those obtained using N- and C-terminal sequences. However, there were also inconsistencies between the terminal region analyses, suggesting that phylogenetic relationships inferred on the basis of fliC sequence should be treated with caution. Although the central domain of fliC is highly variable between Pseudomonas strains, there was evidence of sequence similarities between the central domains of different Pseudomonas fliC sequences. This indicates the possibility of recombination in the central domain of fliC genes within Pseudomonas species, and between these genes and those from other bacteria.     

AlkB homologues in thermophilic bacteria of the genus Geobacillus

Screening of alkane hydroxylase genes (alkB) was performed in the thermophilic aerobic bacteria of the genus Geobacillus. Total DNA was extracted from the biomass of 11 strains grown on the mixture of saturated C10-C20 hydrocarbons, PCR amplification of fragments of alkB genes was performed with degenerate oligonucleotide primers, PCR products were cloned and sequenced. For the first time in the genome of thermophilic bacteria the presence of a set of alkB gene homologues was revealed. The strains each contain three to six homologues among which only two are universal for all of the strains. Comparative phylogenetic analysis of the nucleotide sequences and the inferred amino acid sequences showed close relatedness of six of the revealed variants of geobacilli sequences to the alkB4, alkB3, and alkB2 genes that had previously been revealed by other authors in Rhodococcus erythropolis strains NRRL B-16531 and Q15. The rest two variants of alkB sequences were unique. Analysis of the GC composition of all the Geobacillus alkB homologues revealed closer proximity to the rhodococcal chromosomal DNA than to the chromosomal DNA of geobacilli. This may be an indication of the introduction of the alkB genes into the Geobacillus genome by interspecies horizontal transfer; and rhodococci or other representatives of the Actinobacteria phylum were probably the donors of these genes. Analysis of the codon usage in fragments of alkB genes confirms the suggestion that the pool of these genes is common to the majority of Gram-positive and certain Gram-negative bacteria. Formation of a set of several alkB homologues in a genome of a particular microorganism may result from free gene exchange within this pool.     

A Universal Method for the Identification of Bacteria Based on General PCR Primers

The Universal Method (UM) described here will allow the detection of any bacterial rDNA leading to the identification of that bacterium. The method should allow prompt and accurate identification of bacteria. The principle of the method is simple; when a pure PCR product of the 16S gene is obtained, sequenced, and aligned against bacterial DNA data base, then the bacterium can be identified. Confirmation of identity may follow. In this work, several general 16S primers were designed, mixed and applied successfully against 101 different bacterial isolates. One mixture, the Golden mixture7 (G7) detected all tested isolates (67/67). Other golden mixtures; G11, G10, G12, and G5 were useful as well. The overall sensitivity of the UM was 100% since all 101 isolates were detected yielding intended PCR amplicons. A selected PCR band from each of 40 isolates was sequenced and the bacterium identified to species or genus level using BLAST. The results of the UM were consistent with bacterial identities as validated with other identification methods; cultural, API 20E, API 20NE, or genera and species specific PCR primers. Bacteria identified in the study, covered 34 species distributed among 24 genera. The UM should allow the identification of species, genus, novel species or genera, variations within species, and detection of bacterial DNA in otherwise sterile samples such as blood, cerebrospinal fluid, manufactured products, medical supplies, cosmetics, and other samples. Applicability of the method to identifying members of bacterial communities is discussed. The approach itself can be applied to other taxa such as protists and nematodes.     

Classification of plant associated bacteria using RIF, a computationally derived DNA marker

A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format.     

Unexpectedly diverse Mesorhizobium strains and Rhizobium leguminosarum nodulate native legume genera of New Zealand, while introduced legume weeds are nodulated by Bradyrhizobium species

The New Zealand native legume flora are represented by four genera, Sophora, Carmichaelia, Clianthus, and Montigena. The adventive flora of New Zealand contains several legume species introduced in the 19th century and now established as serious invasive weeds. Until now, nothing has been reported on the identification of the associated rhizobia of native or introduced legumes in New Zealand. The success of the introduced species may be due, at least in part, to the nature of their rhizobial symbioses. This study set out to address this issue by identifying rhizobial strains isolated from species of the four native legume genera and from the introduced weeds: Acacia spp. (wattles), Cytisus scoparius (broom), and Ulex europaeus (gorse). The identities of the isolates and their relationship to known rhizobia were established by comparative analysis of 16S ribosomal DNA, atpD, glnII, and recA gene sequences. Maximum-likelihood analysis of the resultant data partitioned the bacteria into three genera. Most isolates from native legumes aligned with the genus Mesorhizobium, either as members of named species or as putative novel species. The widespread distribution of strains from individual native legume genera across Mesorhizobium spp. contrasts with previous reports implying that bacterial species are specific to limited numbers of legume genera. In addition, four isolates were identified as Rhizobium leguminosarum. In contrast, all sequences from isolates from introduced weeds aligned with Bradyrhizobium species but formed clusters distinct from existing named species. These results show that native legume genera and these introduced legume genera do not have the same rhizobial populations.     

Isolation and characterization of 23 carbofuran-degrading bacteria from soils from distant geographical areas

The aim of this work was to isolate, identify and type carbofuran-degrading bacteria from two geographically distant soils. Restriction Fragment Length Polymorphism (RFLP) patterns of the 16S rRNA gene and partial 16S rRNA sequence analysis were used to classify the 23 isolates obtained. Nine of them showed high similarity to Pseudomonas strains, seven showed similarity to the Flexibacter/Cytophaga/Bacteroides group and the remainder showed similarity to other bacterial genera. Isolates within the same group were sub-typed by comparing partial 16S rRNA sequences and SDS-PAGE analysis of their total protein profiles. Many of the UK isolates showed similarity to the Pseudomonas genera, while most of the Greek isolates showed similarity to the Flexibacter/Cytophaga/Bacteroides group. Only two Chrysobacterium strains isolated from both the UK and Greek soils were identical.     

Diversity and relationships of bradyrhizobia from legumes native to eastern North America

DNA sequencing and polymerase chain reaction (PCR) assays with lineage-specific primers were used to analyze the diversity of 276 isolates of Bradyrhizobium sp. nodule bacteria associated with 13 native legumes species in the northeastern United States, representing eight genera in six legume tribes. A PCR screen with two primer pairs in the rRNA region indicated that seven of the legume species were exclusively associated with strains having markers resembling Bradyrhizobium elkanii, while the remaining six host species harbored strains related to both B. elkanii and Bradyrhizobium japonicum. Sequence analysis of 22 isolates for portions of 16S rRNA and 23S rRNA yielded congruent phylogenetic trees and showed that isolates from different legume genera often shared similar or identical sequences. However, trees inferred from portions of two other genes (alpha-ketoglutarate dioxygenase gene (tfdA), the alpha-subunit of nitrogenase (nifD)) differed significantly from the rRNA phylogeny. Thus, for Bradyrhizobium populations in this region, lateral gene transfer events appear to have altered genealogical relationships of different portions of the genome. These results extend the number of likely cases of gene transfer between divergent taxa of Bradyrhizobium (from members of the B. elkanii lineage to the B. japonicum group) and suggest that transfers have also occurred among separate subgroups of the B. elkanii lineage.     

Production of quorum-sensing-related signal molecules by epiphytic bacteria inhabiting wheat heads

The production of quorum-sensing-related signal molecules (QSRMs) among culturable bacteria comprising the community on wheat heads was investigated. The taxonomic position of 186 bacterial isolates obtained from ten heads was inferred based on 16S rRNA gene sequences, and their QSRM production was determined using two bioreporter strains of N-acylhomoserine lactones. Approximately 33% of isolates produced QSRMs, though the proportion of QSRM-producing isolates on a wheat head was significantly negatively correlated with population size. Most of the producing isolates were Pantoea species, most commonly Pantoea ananatis. Furthermore, the proportion of Pantoea ananatis that produced QSRMs was significantly negatively correlated with the number of bacterial genera (community richness) on each head. Finally, community richness was positively correlated with population size. Qualitative analysis using thin-layer-chromatography revealed that the QSRMs of Pantoea isolates were composed of at least two compounds. This is the first report indicating that Pantoea ananatis isolates inhabiting wheat heads are capable of producing QSRMs. QSRM production by Pantoea spp. may contribute to the predominance of this genus on wheat heads, particularly at relatively low population densities and community diversity.     

Analysis of the 5.8S rRNA gene and the internal transcribed spacers in Naegleria spp. and in N. fowleri

Internal transcribed spacers (ITS) and the 5.8S ribosomal gene of 21 Naegleria fowleri strains and eight other species including Naegleria gruberi were sequenced. The results showed that this region can help differentiate between and within species. The phylogeny of Naegleria spp. deduced from the ITS and the 5.8S gene produced four major lineages, fowleri-lovaniensis, galeacystis-italica-clarki-gruberi-australiensis, andersoni-jamiesoni, and pussardi, that fit perfectly with those inferred from the 18S rRNA gene analysis. The N. gruberi isolate, NG260, was closely related to Naegleria pussardi. The other N. gruberi isolates branched together with Naegleria australiensis in another lineage. The ITS and 5.8S results for N. fowleri were congruent with those previously deduced by RAPD analysis. The phylogenetic analysis inferred from ITS and RAPD data revealed two major groups. The French Cattenom and Chooz and South Pacific strains constituted the first group. The second group encompassed the strains corresponding to the Euro-American and Widespread RAPD variants and shared the same substitution in the 5.8S gene. In addition, it was possible to define species specific primers in ITS regions to rapidly identify N. fowleri.     

Unusual pattern of bacterial ice nucleation gene evolution

Bacterial ice nucleation activity (INA+ phenotype) can be traced to the product of a single gene, ina. A remarkably sparse distribution of this phenotype within three bacterial genera indicates that the ina gene may have followed an unusual evolutionary path. Southern blot analyses, coupled with assays for ice-nucleating ability, revealed that within four bacterial species an ina gene is present in some strains but absent from others. Results of hybridization experiments using DNA fragments that flank the ina gene suggested that the genotypic dimorphism of ina may be anomalous. A phylogenetic analysis of 16S ribosomal RNA gene sequences from a total of 14 ina+ and ina- bacterial strains indicated that the ina+ bacteria are not monophyletic but instead phylogenetically interspersed among ina- bacteria. The relationships of ina+ bacteria inferred from ina sequence did not coincide with those inferred from the 16S data. These results suggest the possibility of horizontal transfer in the evolution of bacterial ina genes.      

Use of an arbitrarily primed PCR product in the development of a Campylobacter jejuni-specific PCR.

Development of a PCR assay for Campylobacter jejuni is based on the isolation of species-specific DNA. An arbitrarily primed PCR incorporating 10-mer primers was used to generate fingerprints of C. jejuni M129 genomic DNA. Fingerprint products were then screened individually for their species specificity in dot blot hybridizations with 6 C. jejuni isolates, 4 Campylobacter species other than C. jejuni, and 27 enteric bacterial species other than Campylobacter spp. A 486-bp fingerprint product hybridized specifically to C. jejuni DNA under stringent conditions; no binding to Campylobacter DNA other than that of C. jejuni or to DNA from enteric bacteria was detected. The 486-bp fingerprint product was sequenced, and primers corresponding to three overlapping regions of the DNA probe were synthesized. Evaluation of the three primer pairs for specificity to C. jejuni DNA identified an oligonucleotide primer pair which amplified a 265-bp product from six C. jejuni isolates only. In sensitivity studies using a crude M129 lysate as the template, the C. jejuni-specific PCR amplified the 265-bp product in a lysate with as few as 100 bacteria.     

用传统分离培养法和高通量测序技术分析印度块菌子囊果内细菌的群落结构

块菌是重要的经济真菌, 在其生长发育过程中, 细菌扮演了重要角色。本文利用传统分离培养方法和高通量测序技术分析了印度块菌(Tuber indicum)子囊果内细菌的群落结构。共分离得到细菌532株, 根据物种累积曲线, 选取其中的112株细菌进行了16S rRNA基因序列分析, 共鉴定出4属40种, 其中假单胞菌属(Pseudomonas)菌株占所测菌株数的80%, 不动杆菌属(Acinetobacter)占12.5%, 链霉菌属(Streptomyces)占5%, 贪噬菌属(Variovorax)占2.5%。通过对印度块菌子囊果16S rRNA基因的V1-V3区高通量测序分析, 共获得细菌序列9,862条, 分属于7门43属220种, 其中变形菌门、拟杆菌门和放线菌门的物种占总物种数的99.7%, 是印度块菌子囊果内的优势细菌。黄杆菌属(Flavobacterium)、壤霉菌属(Agromyces)、微杆菌属(Microbacterium)、剑菌属(Ensifer)和寡养食单胞菌属(Stenotrophomonas)的物种数占总物种的86.3%, 是印度块菌子囊果内细菌的优势属。研究结果表明, 采用胰蛋白大豆培养基仅分离得到印度块菌子囊果内少数细菌物种, 而采用高通量测序技术分析发现, 印度块菌子囊果内细菌物种种类丰富, 群落结构复杂。     

Construction of species-specific primers for Pseudomonas andropogonis based on 16S rDNA sequences

Approximately 94% of the total 16S rDNA of Pseudomonas andropogonis strain ACH 01053A was sequenced and compared with that of strain ATCC 23061 obtained from the GenBank database. The two sequences were highly homologous with 1·3% difference. Alignment of sequences with those from closely related bacteria revealed two possible regions for design of a specific primer suitable for detection of Ps. andropogonis . Using primers designed to these variable regions in a PCR test, an amplification product of approximately 410 bp was specifically produced by 40 strains of Ps. andropogonis . No other bacterial species showed an amplification product under optimized PCR conditions. As few as 1000 cells per reaction were detected.     

Consensus-degenerate hybrid oligonucleotide primers for amplification of priming glycosyltransferase genes of the exopolysaccharide locus in strains of the Lactobacillus casei group

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3' degenerate core based on four highly conserved amino acids and a longer 5' consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS(-)) and EPS-producing (EPS(+)) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS(+) bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS(+) strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.     

油樟内生芽孢细菌的系统发育多样性

摘要：【目的】为了解油樟产芽孢内生细菌的多样性。【方法】采用改良的牛肉膏琼脂培养基分离、去除冗余及芽孢染色,测定所得产芽孢内生细菌的16S rRNA基因,进行系统发育分析。【结果】40株产芽孢内生细菌数量占分离得到的内生细菌总数的38.1％,其中根、茎、叶中分别分离得到24株、7株和9株。16S rRNA基因序列系统发育分析结果表明,35株菌可能分属于Bacillus、Lysinibacillus、Paenibacillus属的16个种,还有5株菌的序列与数据库中典型菌株序列相似性低于97％,代表着潜在新类群的存在。【结论】从油樟3个部位分离出的产芽孢内生细菌存在明显的系统发育多样性,而且3个部位分离出的产芽孢内生细菌区系既呈现出一定程度的细菌区系相似性,又表现出器官细菌区系的特异性。     

A rapid method for identification of typical Leuconostoc species by 16S rDNA PCR-RFLP analysis

A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method was developed to detect and identify typical Leuconostoc species. This method utilises a set of specific primers for amplification of the 16S rDNA region of typical Leuconostoc species. All Leuconostoc-type strains, all Leuconostoc isolates from kimchi, Korea's traditional, fermented vegetable product, and strains from closely related genera were examined to verify the identification by this method. The primers resulted in amplification only for nine typical Leuconostoc spp., but not for any other genera tested. The size of the amplified products was 976 bp and the amplicons of the different species could be differentiated from each other with MseI, HaeIII and Tsp509I endonucleases, except for the species Leuconostoc argentinum and Leuconostoc lactis, which were indistinguishable. A PCR-RFLP method for the typical Leuconostoc species was optimized to identify a large number of isolates from fermented vegetable product. This PCR-RFLP method enables the rapid and reliable identification of Leuconostoc species and to distinguish them from the other phylogenetically related lactic acid bacteria in food samples.     

Cloning, sequencing, and characterization of genomic subtracted sequences from Listeria monocytogenes

Individual sequences of a genomic subtracted, PCR-amplified, mixed-sequence probe (GS probe) were cloned and sequenced. The GS probe differentiated restriction fragment length polymorphism patterns for Listeria monocytogenes but did not hybridize with members of other bacterial genera. Sequence analysis identified several L. monocytogenes sequences already present in the GenBank database; the putative identities of other sequences were inferred from homology data, and still other sequences did not exhibit significant levels of homology with any GenBank sequences.