Phenotypic and genotypic characterization of mutants of the virginiamycin producing strain 899 and its relatedness to the type strain of Streptomyces virginiae.

A representative set of 19 mutants, with a known genealogy, of the virginiamycin producing strain Streptomyces virginiae 899 was investigated phenotypically and genotypically. Colour of the aerial and substrate mycelium were very variable both among spontaneous variants and those obtained after induced mutagenesis. At genotypic level, all mutants showed nearly identical BOX patterns, not reflecting the phenotypic heterogeneity observed. More than 40 years of forced mutational pressure did not cause huge chromosomal distortions but was most likely limited to base substitutions. The species S. virginiae, including besides producers of virginiamycin the type strain and non-type strains producing other bioactive compounds, is genomically heterogeneous on the basis of BOX-PCR fingerprinting and DNA-DNA hybridizations. The virginiamycin producing strain 899 does not belong to the species S. virginiae despite its phenotypic similarity to the latter.

Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice.

The family Acetobacteraceae currently includes three known nitrogen-fixing species, Gluconacetobacter diazotrophicus, G. johannae and G. azotocaptans. In the present study, acetic acid-producing nitrogen-fixing bacteria were isolated from four different wetland rice varieties cultivated in the state of Tamilnadu, India. Most of these isolates were identified as G. diazotrophicus on the basis of their phenotypic characteristics and PCR assays using specific primers for that species. Based on 16S rDNA partial sequence analysis and DNA: DNA reassociation experiments the remaining isolates were identified as Acetobacter peroxydans, another species of the Acetobacteraceae family, thus far never reported as diazotrophic. The presence of nifH genes in A. peroxydans was confirmed by PCR amplification with nifH specific primers. Scope for the findings: This is the first report of the occurrence and association of N2-fixing Gluconacetobacter diazotrophicus and Acetobacter peroxydans with wetland rice varieties. This is the first report of diazotrophic nature of A. peroxydans.

DNA:DNA hybridization and chemotaxonomic studies of Thermus scotoductus.

The species Thermus scotoductus was recently described as containing several non-pigmented isolates from Selfoss, Iceland, and the X-1 strain from the USA (Kristjansson et al., 1994). In this study, we performed DNA:DNA hybridizations and chemotaxonomic studies on several non-pigmented Thermus isolates from other geographical areas to assess their relationship to the strains originally assigned to this species. The results of DNA:DNA hybridizations showed that strains NH and Dl from London and strains Vl-7a and Vl-13 from Vizela, Portugal, belonged to T. scotoductus. T. scotoductus X-1 (ATCC 27978) was composed of two stable colony types, one of which had a major glycolipid different from the one present in the other colony type and from all other Thermus strains examined as well. The fatty acid composition of the isolates from Selfoss and London were practically identical. However, the fatty acid composition of strain X-1, the individual colony types of this strain and the Vizela strains were different from the Selfoss-London isolates and from each other. Another non-pigmented strain, designated SPS-11, belonged to a different DNA homology group.

Search for peptidic molecular markers in hemolymph of crowd-(gregarious) and isolated-reared (solitary) desert locusts, Schistocerca gregaria.

An HPLC analysis of hemolymph extracts was undertaken to uncover differences between desert locusts, Schistocerca gregaria, reared under either crowded or isolated conditions. Some differences in the chromatographic pattern could be detected. One of the major peaks in the hemolymph of crowd-reared adults was found to be a minor one in isolated-reared individuals, whereas other peaks increased after solitarization. The differences became even more pronounced after several generations of isolated rearing. The dominant chromatographic peak in hemolymph extracts of the crowd-reared animals was identified as a novel peptide with a molecular mass of 6080Da. Edman degradation in combination with enzymatic fragmentation and quadrupole-time of flight (Q-Tof) mass spectrometry revealed the full sequence: DNADEDTICVAADNKFYLYANSLKLYTCYNQLPKVYVVKPKSQCRSSLSDCPTS. This 54 aa-peptide is very abundant in hemolymph of crowd-reared adults. Its concentration in hemolymph amounts to 0.1mM. To uncover the function, its effects were investigated in several bioassays, so far without positive results. One of the other peaks differentially expressed in the individuals of the two phases was identified as SGPI-2 (MW=3794Da), which is a serine protease inhibitor in locusts.

A polyphasic approach towards the identification of strains belonging to Lactobacillus acidophilus and related species.

A set of 98 strains belonging to nine species of the Lactobacillus acidophilus rRNA-group have been analysed by SDS-PAGE of cellular proteins, RAPD-PCR and AFLP with fluorescently labeled primers in order to find improved methods for their identification. Strains of the following phenotypically highly similar species were examined: L. acidophilus, L. amylovorus, L. crispatus, L. johnsonii, L. gasseri, L. gallinarum, L. helveticus, L. iners and L. amylolyticus. Although the majority of the species can be differentiated by SDS-PAGE of whole-cell proteins, the latter technique showed poor discrimination between L. gasseri and L. johnsonii strains and between some strains of L. amylovorus and L. gallinarum. However, this study shows that the RAPD-PCR (using at least 3 different primers followed by numerical analysis of the combined patterns) and AFLP are most suitable genomic fingerprinting techniques for the differentiation of all the species listed above, and that databases for identification can be constructed, particularly when commercially available molecular tool-kits are used. The separate species status of the recently described L. amylolyticus and L. iners was fully confirmed.

Genomic diversity amongst Vibrio isolates from different sources determined by fluorescent amplified fragment length polymorphism.

The genomic diversity among 506 strains of the family Vibrionaceae was analysed using Fluorescent Amplified Fragments Length Polymorphisms (FAFLP). Isolates were from different sources (e.g. fish, mollusc, shrimp, rotifers, artemia, and their culture water) in different countries, mainly from the aquacultural environment. Clustering of the FAFLP band patterns resulted in 69 clusters. A majority of the actually known species of the family Vibrionaceae formed separate clusters. Certain species e.g. V. alginolyticus, V. cholerae, V. cincinnatiensis, V. diabolicus, V. diazotrophicus, V. harveyi, V. logei, V. natriegens, V. nereis, V. splendidus and V. tubiashii were found to be ubiquitous, whereas V. halioticoli, V. ichthyoenteri, V. pectenicida and V. wodanis appear to be exclusively associated with a particular host or geographical region. Three main categories of isolates could be distinguished: (1) isolates with genomes related (i.e. with > or =45% FAFLP pattern similarity) to one of the known type strains; (2) isolates clustering (> or =45% pattern similarity) with more than one type strain; (3) isolates with genomes unrelated (<45% pattern similarity) to any of the type strains. The latter group consisted of 236 isolates distributed in 31 clusters indicating that many culturable taxa of the Vibrionaceae remain as yet to be described.

Differences between subcultures of the Mesorhizobium loti type strain from different culture collections.

Because of differences in the reported 16S rRNA gene sequence of the Mesorhizobium loti type strain available from different culture collections, we collected different subcultures of this strain and compared them by 16S rDNA sequencing, SDS-PAGE of whole-cell protein extracts and RAPD-PCR. Our results indicate that the 16S rDNA sequence differences can be explained by the presence of two different organisms in one of the subcultures. In addition, even for subcultures of the type strain that had identical 16S rDNA sequences, small differences could be observed in the protein profiles and in the RAPD-PCR patterns. These latter observations indicate that maintenance procedures necessary for long-term preservation by freeze-drying can cause subcultures of the same original strain to undergo changes, effectively leading to different fingerprints even though 16S rDNA sequences remain identical.

AFLP fingerprint analysis of Bradyrhizobium strains isolated from Faidherbia albida and Aeschynomene species.

The diversity of Bradyrhizobium isolates from Faidherbia albida and Aeschynomenee species was assessed using AFLP analysis, a high-resolution genomic fingerprinting technique. Reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii and Bradyrhizobium liaoningense were included for comparison. At a similarity level of 50%, a total of 34 different groups were obtained by cluster analysis of the genomic fingerprints. Four of these clusters correspond to the three reference species, demonstrating the large diversity of the isolates studied. Comparison with other data demonstrates that AFLP has a higher resolution than restriction analysis of 16S rRNA genes, SDS-PAGE analysis of proteins and phenotypic analysis. Results of the latter two methods showed little correspondence with the genotypic data.

The coral bleaching Vibrio shiloi Kushmaro et al. 2001 is a later synonym of Vibrio mediterranei Pujalte and Garay 1986.

The coral bleaching Vibrio shiloi LMG 19703T was characterized by means of Fluorescent Amplified Fragment Length Polymorphism (FAFLP), DNA-DNA hybridisation, mol% G+C content, fatty acids methyl ester (FAME) analysis and phenotypical tests. Numerical analysis of the FAFLP band patterns indicated that the type strain of V. shiloi in fact belongs to the species V. mediterranei. The type strains of both species shared 77% DNA similarity, as determined by DNA-DNA hybridisation experiments at stringent conditions. Moreover, V. shiloi and V. mediterranei showed almost identical fatty acid composition and phenotypical features. Collectively, the genotypic and phenotypic data presented in this study suggest that V. shiloi Kushmaro et al. 2001 should be considered a later synonym of V. mediterranei Pujalte and Garay 1986. The involvement of V. mediterranei in coral bleaching was unknown until now.

Differentiation of Lactobacillus plantarum, L. pentosus and L. paraplantarum species by RAPD-PCR and AFLP.

Two high-resolution genotypic techniques (RAPD-PCR and AFLP) were evaluated for their possibility to discriminate the species Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum and to type these taxa at the infra-species level. In total 23 strains of L. plantarum, three strains of L. pentosus, two strains of L. paraplantarum and two related strains for which the species assignment was not clear, were studied. For RAPD-PCR, suitable oligonucleotides and amplification conditions were selected and tested. For AFLP, a double digest of total genomic DNA was used and a subset of restriction fragments was selectively amplified and visualised using different primer combinations. Both methodologies generated, species-specific electrophoretic profiles. Moreover, the presence of distinct subgroups was revealed within the species L. plantarum.

Polyphasic characterization of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (p(HB-co-HV)) metabolizing and denitrifying Acidovorax sp. strains.

For the purpose of denitrification in small drinking water plants, a bacterial mixed population was isolated from a packed bed column bioreactor with poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P(HB-co-HV)) as a substrate for the denitrification of ground water (10 degrees C). Isolates 2nIII from the mixed culture, with the ability to denitrify and metabolize P(HB-co-HV), were used as starter cultures for the elimination of nitrate in ground water. The strains were characterized by diverse techniques. Classical phenotypic studies lead to rRNA group III of the genus Pseudomonas. Results obtained by molecular techniques demonstrated that the 2nIII strains are members of the Comamonadaceae and shows similarities to the genus Acidovorax. However, an integration of the 2nIII isolates within one of the known Acidovorax species is not possible for the moment. The 2nIII starter cultures clustered close to Av. temperans according to their whole cell proteins and fatty acids, whereas in DNA/DNA hybridization no significant DNA binding (< 25%) was found. In contrast a significant but low degree of DNA/DNA hybridization was found between the 2nIII strains and Av. facilis and Av. delafieldii. Our polyphasic results lead to the conclusion that the 2nIII strains may constitute a separate Acicdovorax species.

The lactobin A and amylovorin L471 encoding genes are identical, and their distribution seems to be restricted to the species Lactobacillus amylovorus that is of interest for cereal fermentations.

Lactobin A and amylovorin L471 are two bacteriocins produced by the phenotypically different strains Lactobacillus amylovorus LMG P-13139 and L. amylovorus DCE 471, respectively. A 110-bp PCR fragment of the structural gene of lactobin A was obtained from total genomic DNA of L. amylovorus LMG P-13139, which was used as a probe to isolate a 3.6-kb HindIII chromosomal fragment for sequencing. PCR amplification revealed that both the structural genes of both the bacteriocins lactobin A and amylovorin L471 were identical. These bacteriocins will be further referred to as amylovorin L. Amylovorin L can be defined as a small, strongly hydrophobic, antibacterial peptide consisting of 50 amino acids. It is synthesized as a precursor peptide of 65 amino acids processed at a characteristic double-glycine proteolytic cleavage site. Amylovorin L hence belongs to the class II bacteriocins. It has a narrow inhibitory spectrum, being most active towards Lactobacillus delbrueckii subsp. bulgaricus LMG 6901(T). Among 38 strains of the Lactobacillus acidophilus DNA homology group, another 6 L. amylovorus strains were also inhibitory towards the L. delbrueckii subsp. bulgaricus LMG 6901(T) strain. The lactobin A or amylovorin L471 structural genes could be detected in the genomes of three of these L. amylovorus strains, but only after extensive PCR amplification, indicating that the inhibitory substances were slightly different. The bacteriocins were characterized as small (approximately 4800 Da), heat-stable peptides that were active in a wide pH range (2.2-8.0). Finally, preliminary experiments indicated that the production of amylovorin L by L. amylovorus DCE 471 took place during a natural rye fermentation, indicating its potential importance in the development of a functional (probiotic) starter culture for cereal fermentations.

Simultaneous typing of C3 and Tf and of Gc and Bf on cellogel electrophoresis.

A method is described to type C3 and Tf on the same Cellogel strip (and Bf simultaneously on a parallel strip). A different method allows to type Gc and Bf on one Cellogel strip. These methods are rapid, easy and reliable, although agarose electrophoresis is a little more effective.

Application of a Y-STR-pentaplex PCR (DYS19, DYS389I and II, DYS390 and DYS393) to sexual assault cases.

A survey of 89 random rape cases including 166 semen traces, according to the prostate-specific-antigen test, showed the need for a more efficient method of DNA-profiling. No male autosomal STR-profile could be detected, after differential lysis, in 35% of the 84 samples usually containing high amounts of the victim's cells, i.e. vaginal/anal washings and knickers. A Y-STR-pentaplex was applied to the stored sperm fraction of 50 unsolved semen traces, with success in 48% of them. The chance of success depends of the amount of total DNA available and of the quality of the extracted DNA.

Results of a collaborative study of the EDNAP group regarding the reproducibility and robustness of the Y-chromosome STRs DYS19, DYS389 I and II, DYS390 and DYS393 in a PCR pentaplex format.

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in the frame work of the STADNAP program, i.e. standardization of DNA profiling in Europe, in order to evaluate the performance of a Y-chromosome STR pentaplex, which includes the loci DYS19, DYS389 I and II, DYS390 and DYS393 and to determine whether uniformity of results could be achieved among different European laboratories. Laboratories were asked to analyze the five Y-STRs using singleplex and multiplex conditions in three bloodstains and one mixed stain (95% female and 5% male). All the laboratories reported the same results even for the mixed stain included in the exercise. This demonstrates the reproducibility and robustness of Y-chromosome STR typing even with multiplex formats and proves the usefulness of Y-STR systems for analyzing mixed stains with a male component.A total of 930 male samples from 10 different populations from Europe were also analysed for all the loci included in the pentaplex. Eight of these ten populations also included haplotype data. As for single gene analysis, haplotype diversity was higher in Germany and Italy and lower in Western European countries and Finland. Pairwise haplotype analysis shows the Finnish departure from the rest of the populations and a relatively homogeneity in the other European populations with F(ST) estimates lower than 0.05.UPGMA analysis shows an association of Western European population (Ireland, UK, Portugal and Galicia) on the one hand and central European populations on the other.

Online reference database of European Y-chromosomal short tandem repeat (STR) haplotypes.

The reference database of highly informative Y-chromosomal short tandem repeat (STR) haplotypes (YHRD), available online at http://ystr.charite.de, represents the largest collection of male-specific genetic profiles currently available for European populations. By September 2000, YHRD contained 4688 9-locus (so-called "minimal") haplotypes, 40% of which have been extended further to include two additional loci. Establishment of YHRD has been facilitated by the joint efforts of 31 forensic and anthropological institutions. All contributing laboratories have agreed to standardize their Y-STR haplotyping protocols and to participate in a quality assurance exercise prior to the inclusion of any data. In view of its collaborative character, and in order to put YHRD to its intended use, viz. the support of forensic caseworkers in their routine decision-making process, the database has been made publicly available via the Internet in February 2000. Online searches for complete or partial Y-STR haplotypes from evidentiary or non-probative material can be performed on a non-commercial basis, and yield observed haplotype counts as well as extrapolated population frequency estimates. In addition, the YHRD website provides information about the quality control test, genotyping protocols, haplotype formats and informativity, population genetic analysis, literature references, and a list of contact addresses of the contributing laboratories.

Diversity of Shewanella population in fish Sparus aurata harvested in the Aegean Sea.

AIMS: To study the diversity of Shewanella population in Sparus aurata fish harvested in the Aegean Sea, as well as to elucidate the influence of fish storage conditions on the selection in Shewanella strains. METHODS AND RESULTS: A total of 108 strains of Shewanella spp. were isolated from Sparus aurata during storage under various conditions. Conventional phenotypic analysis along with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell proteins and 16S rRNA sequence analysis were used for the characterization of the strains. Numerical analysis of whole cell protein profiles showed that the isolates were separated into two distinct clusters A and B with 47% similarity. Cluster B was further subdivided into two subclusters B1 and B2 with 70% similarity. One strain could not be assigned to any of these groups. The different ability of isolates to utilize deoxycholate, D-saccharate, D-glucuronate, N-acetyl-glycosamine, D-maltose, gluconate and citrate, as well as the different type of metabolism on the Hugh and Leifson medium distinguished the different Shewanella biogroups, as these were defined by the SDS-PAGE analysis. Representative strains from the three biogroups were further investigated by 16S rRNA sequence analysis and showed more than 99.4% similarity. CONCLUSIONS: Significant similarities between the isolates and the type strains of S. baltica, S. putrefaciens and S. oneidensis at both phenotypic and molecular level signalize that the new isolates are closely related with the above Shewanella species, but do not provide a clear evidence to which of these species they belong. SIGNIFICANCE AND IMPACT OF THE STUDY: The lack of information about the diversity of Shewanella population in Sparus aurata fish originated from Mediterranean Sea could be confronted using conventional phenotypic techniques, SDS-PAGE analysis of whole cell proteins and 16S rRNA sequencing.

Sources of the adventitious microflora of a smear-ripened cheese.

AIMS: To determine the relationships between the major organisms from the cheese-making personnel and environment and the surface of a smear cheese. METHODS AND RESULTS: 360 yeast and 593 bacteria from the cheese surface, the dairy environment and the hands and arms of personnel were collected. Pulsed-field gel electrophoresis, repetitive sequence-based polymerase chain reaction and 16S rDNA sequencing were used for typing and identifying the bacteria, and mitochondrial DNA restriction fragment length polymorphism and Fourier-transform infrared spectroscopy for typing and identifying the yeast. The three most dominant bacteria were Corynebacterium casei, Corynebacterium variabile and Staphylococcus saprophyticus, which were divided into three, five and seven clusters, respectively, by macrorestriction analysis. The same clones from these organisms were isolated on the cheese surface, the dairy environment and the skin of the cheese personnel. Debaryomyces hansenii was the most dominant yeast. CONCLUSIONS: A 'house' microflora exists in the cheese plant. Although the original source of the micro-organisms was not identified, the brines were an important source of S. saprophyticus and D. hansenii and, additionally, the arms and hands of the workers the sources of C. casei and C. variabile. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the major contribution of the house microflora to the ripening of a smear-ripened cheese has been demonstrated.

Photobacterium rosenbergii sp. nov. and Enterovibrio coralii sp. nov., vibrios associated with coral bleaching.

Six new Vibrio-like isolates originating from different species of bleached and healthy corals around Magnetic Island (Australia) were investigated using a polyphasic approach. Phylogenetic analyses based on 16S rRNA, recA and rpoA gene sequences split the isolates in two new groups. Strains LMG 22223(T), LMG 22224, LMG 22225, LMG 22226 and LMG 22227 were phylogenetic neighbours of Photobacterium leiognathi LMG 4228(T) (95.6 % 16S rRNA gene sequence similarity), whereas strain LMG 22228(T) was related to Enterovibrio norvegicus LMG 19839(T) (95.5 % 16S rRNA gene sequence similarity). The two new groups can be distinguished from closely related species on the basis of several phenotypic features, including fermentation of d-mannitol, melibiose and sucrose, and utilization of different compounds as carbon sources, arginine dihydrolase activity, nitrate reduction, resistance to the vibriostatic agent O/129 and the presence of fatty acids 15 : 0 iso and 17 : 0 iso. The names Photobacterium rosenbergii sp. nov. (type strain LMG 22223(T)=CBMAI 622(T)=CC1(T)) and Enterovibrio coralii sp. nov. (type strain LMG 22228(T)=CBMAI 623(T)=CC17(T)) are proposed to accommodate these new isolates. The G+C contents of the DNA of the two type strains are respectively 47.6 and 48.2 mol%.