A new method for multilocus enzyme electrophoresis, based on electrophoretic transfers to nitrocellulose after polyacrylamide-agarose gel electrophoresis was explored. Electrophoretic separation was performed on 1-mm-thick slab gels with 6-mul samples of bacterial extracts and was followed by serial 5-min consecutive transfers. The transferability of 19 metabolic enzymes of Klebsiella strains was studied and allowed the simultaneous examination of one enzyme in the separation gel and at least five enzymes on nitrocellulose sheets. The resolution of enzyme bands was increased on nitrocellulose; thus, well-separated bands were recorded for nucleoside phosphorylase, peptidase, and phosphoglucose isomerase whereas their mobility variants could not be clearly distinguished in the separation gel because of stain diffusion. The study of genetic relationships of 42 strains of Klebsiella pneumoniae and 24 strains of Klebsiella oxytoca demonstrated the reliability of the method, since clustering analysis of electrophoretic types, based on electrophoretic polymorphism of 10 metabolic enzymes, showed two main clusters well correlated with the two species. The 57 electrophoretic types described confirm the usefulness of the method for the study of genetic relationships between closely related strains.

Esterase (Est) and esterase-D (Est-D) electrophoretic patterns identified by starch gel electrophoresis of skeletal muscle protein extracts of 184 specimens of three species of peacock bass, locally known as tucunares (Cichla monoculus, C. temensis and Cichla sp), plus four specimens of a supposed hybrid (C. monoculus vs C. temensis), collected from the Central Amazon, were examined to determine if they could aid in identifying a supposed hybrid between C. monoculus and C. temensis. Six zones of electrophoretic activity were found with these enzyme systems. The Est enzyme showed one zone of activity, formed by bands 1, 2 and 3, plus three zones of activity, presumably controlled by Est-1, 2 and 3 loci. The Est-D enzyme had two zones of activity, presumably controlled by Est-D1 and Est-D2 loci. Cichla monoculus and C. temensis shared band 2 and alleles Est-1(1), Est-2(1), Est-3(2), and Est-D1(1), and therefore these were useless for identifying hybrids between the two species. However, a probable hybrid pattern of bands 1, 2, and 3, presumably generated by a combination of pattern 12 from C. monoculus with pattern 23 from C. temensis, resulting from a possible cross between these two species, was detected. Although the Est-D2 locus cannot be considered an ideal diagnostic marker for identifying the supposed hybrid (C. monoculus vs C. temensis), as it is polymorphic, it proved to be useful for determining the origin of the hybrid, i.e., which parental species were involved in the hybridization process.

AIMS: To analyse Neisseria meningitidis isolates from meningococcal meningitis cases in Rio de Janeiro (Brazil) from 1990 to 1993 and 1999-2002, to determine the genetic and relatedness with hypervirulent and epidemic strains. METHODS AND RESULTS: The isolates were analysed by multilocus enzyme electrophoresis (MEE) clustering into 83 electrophoretic types (ET). All isolates from 1999 to 2002, formed a cluster which included one strain of the ET-5 complex worldwide associated with epidemics. CONCLUSIONS: The overall results suggested a panmictic structure probably because of recombination events. The observation of a separated cluster including isolates from 1999 to 2002 and an ET-5 complex strain, also suggested the introduction of strains genetically related with this hypervirulent complex in the State of Rio de Janeiro (Brazil) over the last 5 years. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of strains related to the ET-5 complex in several states of Brazil was already described elsewhere, but this is the first time it was reported in the State of Rio de Janeiro. Our findings reinforce the necessity to genetically determine the clones which should be considered to produce a national vaccine against meningococcal meningitis.

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.

Genetic diversity, genetic relationship, identification and population structure of 120 Aeromonas strains (including Aer. hydrophila, Aer. bestiarum, Aer. salmonicida and Aer. popoffii) isolated from various sources were studied by analysis of 15 genetic loci by multilocus enzyme electrophoresis (MLEE). All 15 loci were polymorphic, with an average of 9.4 alleles per locus and a mean genetic diversity (H) of 0.64. Cluster analysis defined at H </= 0.7 differentiated most of the taxa analysed except the Aer. popoffii and Aer. bestiarum strains, which showed a close genetic relationship. Allelic frequencies of five loci (EST1, HEX, IDH, LDH1 and MDH) identified 94% of the strains. The index of association (IA) for the total sample was 2.38 and IA values calculated for the different populations were always significantly different from zero. These results suggest that the population structure of this Aeromonas sample is strongly clonal, confirm the taxonomic status of the analysed species in population genetics terms, and show the usefulness of MLEE for identifying Aeromonas species.

The polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPD) assay, morphological, physiological, biochemical and antimicrobial susceptibility test methods have been evaluated for use in the taxonomy of isolated thermotolerant Bacillus from Jordanian hot springs, with specific reference to strains Geobacillus stearothermophilus (ATCC 12980), Bacillus circulans (ATCC 4513) and Bacillus sphaericus (ATCC 14577). A RAPD assay has been optimized and is able to discriminate between numerous thermotolerant Bacillus strains. RAPD-PCR was found to give reproducible thermotolerant Bacillus strains classification of DNA fingerprints for 14 strains including 3 reference strains. A study of 14 isolates and 3 reference strains, analyzing 53 phenotypic characters, resulted in their allocation to five major clusters at 60% similarity. Whereas at 80% similarity, twelve taxonomically distinct groups were evident.

Monokaryon strains H2 and J3 were respectively developed from the cultivated strains He-1 and Ju-1 of Auricularia auricula through protoplasted monokaryon technique. Dicaryon strain H2J3 was bred through crossbreeding of H2 and J3 as parents. Fifty-two F1 monokaryon strains were obtained from the fruitbodies of dicaryon H2J3 of A. auricula by the single-spore isolation and culture. All the tested strains, parent monokaryon strains (H2,J3) and fifty-two F1 monokaryon strains, were cultured in liquid Complete Yeast Medium(CYM) for twenty days, and the mycelia of tested strains were respectively analysed by polyacrylamide gel electrophoresis. Bian Y B et al. have reported the results of isozyme zymogram polymorphisms of cultured strains including He-1 and Ju-1, and the specific expression of esterase genes on the different stages of mycelial development in A. auricula. The same named methods of isozyme loci and alleles suggested by Gottlieb (1973) were used in this study. The relative fitness of allele segregation and theoretical expected ratio was examined by the contingency chi 2 test. The linkage relationship was assessed according to the method suggested by Rudin and Ekberg (1978). The isozyme analysis of tested strains showed that the isozymes of esterase (EST), malate dehydrogenase(MDH) and formate dehydrogenase(FDH) of A. auricula were respectively controlled by 7, 5 and 4 polymorphic loci. The isozyme bands which were controlled by EST-3, EST-4, MDH-3 and MDH-4 loci were stably expressed in the parental strains and most tested F1 monokaryon strains. Genetic analysis was impossible for these four loci. The relative fitness between allele observed value and theoretical expected value was further examined by means of contingency chi 2 at 1% level, the result indicated that significant difference existed from the chi 2 value of MDH-5 and FDH-1 in 7 polymorphic loci of EST, MDH and FDH, and denied the hypothesis in which the isozyme bands controlled by MDH-5 and FDH-1 loci were presumed to be allozyme bands. The allele observed values (100:SA) of EST-7, MDH-1, MDH-5, FDH-1, FDH-2 and FDH-4 were significant deviation of theoretical expected ratio (1:1), which showed that these variations were not due to the allele expressions. The significant difference was not be showed at the 5% level from the chi 2 values of EST-1, EST-2, EST-5, EST-6 and MDH-2, this result indicated the allele segregation was corresponding to the theoretical expected ratio at these 5 loci, these 5 loci were allozyme loci which was corresponding to Mendel's law of segregation. A total of 10 possible pair combinations from the 5 allozyme loci were tested. The numbers of monokaryon strains with parental type zymogram or recombinant type zymogram were counted up, and the chi 2I, chi 2II and chi 2L were calculated for examining the linkage relationships of these allozyme loci, the chi 2L value attained to significant difference at 1% level between EST-5 and EST-6 loci, it indicated that the linkage relationship existed between the two loci, non-linkage relationships existed between the other pairing loci of 5 allozyme loci.

Nondenaturing polyacrylamide gel electrophoresis revealed the presence of diversity among bacteriocins produced by strains of Bacillus sphaericus. Bacteriocin bands of six strains (pathogenic and non pathogenic) were found to be located just below the stacking gel. However, in two other strains (1 pathogenic and 1 collection strain) more than one protein band with bacteriocin activity were seen in the middle of resolving gel. In bacteriocin-treated cultures, electron-microscopy studies revealed the growth of lysedswollen ghost cells, and loss of viability among sensitive strains.

Triatoma barberi, T. dimidiata, T. longipennis, T. pallidipennis and T. picturata, all key Chagas disease vectors in Mexico, were analysed by multilocus enzyme electrophoresis (MLEE) at 17 putative loci. The majority of insect specimens studied were collected from domestic and peridomestic structures from multiple geographic locations while others were collected from sylvatic areas. T. barberi was the least polymorphic species (P(0.95)=0.18), with polymorphism rates of the other species ranging from 0.29 to 0.50. T. barberi, a member of the protracta complex, clustered apart from the other studied species by Nei's genetic distance with >1.36, and at least eight loci were found to be diagnostic for this species. T. dimidiata was more related to T. longipennis, T. pallidipennis and T. picturata (phyllosoma complex) than to T. barberi, with a genetic distance averaging 0.36 with the phyllosoma complex species. In contrast, the genetic distances between the three phyllosoma complex species were not significantly different from zero, and there were no species-specific loci differentiating among them. The results strongly support the grouping of these three species in one complex, separate from the two other species studied.

Thirty-one urease-positive thermophilic Campylobacter (UPTC) isolates, including three reference strains (NCTC12892, NCTC12895 and NCTC12896), and three Campylobacter lari isolates, which were isolated from several countries and sources, were compared genotypically by using multilocus enzyme electrophoresis (MLEE). We examined allelic variation around seven enzyme loci, including the adenylate kinase, alkaline phosphatase, catalase, fumarase, malic enzyme, malate dehydrogenase, and L-phenylalanyl-L-leucine peptidase loci. MLEE typing revealed the presence of 23 different electrophoretic types (ETs) among the 31 UPTC isolates, and 14 isolates shared six electrophoretic profiles. Three different ETs were identified for the three C. lari isolates examined, and no ETs were shared by UPTC and C. lari isolates. Quantitative analyses were subsequently performed by using allelic variation data, and the results demonstrated that the mean genetic diversity was 0.655. In conclusion, MLEE demonstrated that the UPTC isolates examined are genetically hypervariable and form a cluster separate from the C. lari cluster.

The genetic structure of eight locust species in three families (Catantopidae, Oedipodidae and Arcypteridae) from Shanxi Province in China was compared using allozyme analysis with horizontal starch gel electrophoresis. Among 17 loci identified in zymograms, Ao-1, Est-3, G3pd-1, Idh-2 and Mdh-2 had low variability with a few alleles. High polymorphism was observed at Ldh-1, Me-1 and Gpi-1. Each of the eight species demonstrated high percentage of polymorphic loci (P = 64.7%-94.1%) but low observed heterozygosity (H0 = 0.024-0.087) due to heterozygote deficiency. It was noted that the migratory locusts usually had higher percentage of polymorphic loci (P = 88.2%-94.1%) than non-migratory species (P = 64.7%-94.1%). The only exception is Oxya chinensis(P = 94.1%). It is reasoned that the higher polymorphism is necessary for migratory species to cope with the environments that might be drastically different from the habitats before migration. The taxon relationships using cluster analysis based on Nei's genetic identity (I) and Roger's genetic distance (D) were the same at species and genus levels. The differences were found at family level, possibly due to the alternative algorithms. The cladogram using Roger's genetic distance (D) overlapped the relationship obtained from karyotypic analyses, which demonstrated that the species examined in Catantopidae displayed somewhat closer relationship to those in Oedipodidae than to those in Arcypteridae. It is suggested that the allozyme analysis is useful as molecular marker for locusts in phylogenetic reconstruction at the species and genus level, while additional data from other studies are necessary when used for higher taxa.

Forty-two strains representing the eight recognized nitrogen-fixing Paenibacillus species and 12 non-identified strains were examined by restriction fragment length polymorphism (RFLP) analysis of part of 16S and 23S rRNA genes amplified by polymerase chain reaction (PCR). Eleven different 16S rDNA genotypes were obtained from the combined data of RFLP analysis with four endonucleases and they were in agreement with the established taxonomic classification. Only one group of unclassified strains (Group I) was assigned in a separate genotype, suggesting they belong to a new species. Using the 23S PCR-RFLP method only six genotypes were detected, showing that this method is less discriminative than the 16S PCR-RFLP. Using the multilocus enzyme electrophoresis (MLEE) assay, the 48 strains tested could be classified into 35 zymovars. The seven enzymatic loci tested were polymorphic and the different profiles obtained among strains allowed the grouping of strains into 10 clusters. The PCR-RFLP methods together with the MLEE assay provide a rapid tool for the characterization and the establishment of the taxonomic position of isolates belonging to this nitrogen-fixing group, which shows a great potentiality in promoting plant growth.

A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase was isolated in Port Dickson, Malaysia. The gene for the lipase was recovered from a genomic library and sequenced. Phylogenetic analysis was performed based on an alignment of thirteen microbial lipase sequences obtained from the NCBI database. The analysis suggested that the B. sphaericus lipase gene is a novel gene, as it is distinct from other lipase genes in Families I.4 and I.5 reported so far. Expression in Escherichia coli under the control of the lacZ promoter resulted in an eight-fold increase in enzyme activity after a 3-h induction with 1 mM IPTG. The crude enzyme thus obtained showed a slight (10%) enhancement in activity after a 30-min incubation in 25%(v/v) n-hexane at 37 degrees C, and retained 90% of its activity after a similar period in 25%(v/v) p-xylene.

A PCR system for studying the diversity of species of Bacillus and related taxa directly from soil was developed. For this purpose, a specific 24-bp forward primer located around position 110 of the 16S ribosomal RNA gene was designed and combined with a reverse bacterial primer located at the end of the gene. The specificity of this PCR system for bacilli and related taxons was confirmed on the basis of tests with diverse strains as well as with soil DNA. Analysis of a soil DNA derived clone library showed that the amplified fragments affiliated exclusively with sequences of gram-positive bacteria, with up to 95% of the sequences originating from putative Bacillus species. In particular, sequences affiliated to those of B. mycoides, B. pumilus, B. megaterium, B. thuringiensis, and B. firmus, as well as to related taxa such as Paenibacillus, were obtained. A minority, i.e., less than 6%, of the clones affiliated with other gram-positive bacteria, such as Arthrobacter spp., Frankia spp., and uncultured gram-positives. The amplified fragments were used as templates for a second PCR using bacterial 16S rDNA primers, yielding PCR products of about 410 bp, which were separated by denaturing gradient gel electrophoresis (DGGE). Amplicons indicating Bacillus spp. were found in the gel between 45% and roughly 60% denaturant, whereas those representing other, high-G+C% bacteria, were localized in gel regions with denaturant concentrations exceeding about 60%, thus allowing the distinction between these two groups of sequences. We applied this system to compare the group-specific diversity in bacterial communities in an agricultural soil under different regimes, i.e., permanent grassland, grassland recently turned to arable land, and arable land under agricultural rotation. Differences in the Bacillus-related community structures between the treatments were clearly detected. Higher diversities, as judged by Shannon-Weaver indices calculated on the basis of the molecular profiles, were consistently observed in the permanent grassland and the grassland turned into arable land, as compared to the arable land.

The predominantly selfing slug species Arion (Carinarion) fasciatus, A.(C.) silvaticus and A.(C.) circumscriptus are native in Europe and have been introduced into North America, where each species consists of a single, homozygous multilocus genotype (strain), as defined by starch gel electrophoresis (SGE) of allozymes. In Europe, the "one strain per species" hypothesis does not hold since polyacrylamide gel electrophoresis (PAGE) of allozymes uncovered 46 strains divided over the three species. However, electrophoretic techniques may differ in their ability to detect allozyme variation. Therefore, several Carinarion populations from both continents were screened by applying the two techniques simultaneously on the same individual slugs and enzyme loci. SGE and PAGE yielded exactly the same results, so that the different degree of variation in North American and European populations cannot be attributed to differences in resolving power between SGE and PAGE. We found four A.(C.) silvaticus strains in North America indicating that in this region the "one strain per species" hypothesis also cannot be maintained. Hence, the discrepancies between previous electrophoretic studies on Carinarion are most likely due to sampling artefacts and possible founder effects.

Genetic variation of Contracaecum ogmorhini (sensu lato) populations from different otariid seals of the northern and southern hemisphere was studied on the basis of 18 enzyme loci as well as preliminary sequence analysis of the mitochondrial cyt b gene (260 bp). Samples were collected from Zalophus californianus in the boreal region and from Arctocephalus pusillus pusillus, A. pusillus doriferus and A. australis from the austral region. Marked genetic heterogeneity was found between C. ogmorhini (sensu lato) samples from the boreal and austral region, respectively. Two loci (Mdh-2 and NADHdh) showed fixed differences and a further three loci (Iddh, Mdh-1 and 6Pgdh) were highly differentiated between boreal and austral samples. Their average genetic distance was D(Nei)= 0.36 at isozyme level. At mitochondrial DNA level, an average proportion of nucleotide substitution of 3.7% was observed. These findings support the existence of two distinct sibling species, for which the names C. ogmorhini (sensu stricto) and C. margolisi n. sp., respectively, for the austral and boreal taxon, are proposed. A description for C. margolisi n. sp. is provided. No diagnostic morphological characters have so far been detected; on the other hand, two enzyme loci, Mdh-2 and NADHdh, fully diagnostic between the two species, can be used for the routine identification of males, females and larval stages. Mirounga leonina was found to host C. ogmorhini (s.s.) in mixed infections with C. osculatum (s.l.)(of which C. ogmorhini (s.l.) was in the past considered to be a synonym) and C. miroungae; no hybrid genotypes were found, confirming the reproductive isolation of these three anisakid species. The hosts and geographical range so far recorded for C. margolisi n. sp. and C. ogmorhini (s.s.) are given.

From the Vero Beach strain of the mosquito Aedes (Stegomyia) aegypti (L.)(Diptera: Culicidae), substrains were selected for susceptibility (SS) and refractoriness (RR) to the dog heartworm Dirofilaria immitis (Leidy)(Filarioidea: Onchocercidae). These two lines and their reciprocal F1 hybrids were analysed for genetic variation at 14 enzyme loci, using polyacrylamide gel electrophoresis. Six of the enzyme loci showed variation (sample size 48 alleles/locus/line). Three of these were monomorphic in the refractory line but polymorphic in the susceptible, i.e. aconitase hydratase (Acoh), isocitrate dehydrogenase-1 (Idh-1) and phosphoglucomutase (Pgm). The other three loci, glucose-6-phosphate isomerase (Gpi), hexokinase-1 (Hk-1) and isocitrate dehydrogenase-2 (Idh-2), were polymorphic in both SS and RR lines and their hybrids. At two loci (Hk-1, Pgm) three alleles were detected, whereas the other polymorphic loci had only two alleles. For Hk-1, the most frequent allele was Hk-1(80)(0.563) in refractory and Hk-1(100) in the susceptible (0.521) and F1 hybrids. For Pgm the most frequent alleles were Pgm125 in the susceptible line (0.646) and Pgm100 in the F1 hybrids (0.563 and 0.604) and refractory line (1.000). The mean observed heterozygosity (Ho), the mean Hardy-Weinberg expected heterozygosity (He) and the mean number of alleles per locus in the refractory line were lower, but not significantly so, than in the susceptible line and their reciprocal F1 hybrids; the proportion of polymorphic loci was significantly lower in the refractory than in the susceptible line and their F1 hybrids. Within both lines all polymorphisms were in Hardy-Weinberg equilibrium, whereas significant departures from predicted frequencies were observed in SS x RR hybrids at four polymorphic loci (Acoh, Gpi, Hk-1, Pgm) and at three polymorphic loci (Acoh, Hk-1, Pgm) in RR x SS hybrids. The average Nei's and modified Rogers' genetic distances between the lines were 0.024 and 0.139, respectively. These electrophoretic data show that the refractory line (putatively lacking fi allele) can be distinguished from the susceptible line (fi/fi) and their hybrids (heterozygous fi) by isozyme marker frequencies, but it remains to be seen whether this difference is causal or chance linkage. In any case, this model system of Ae. aegypti/D. immitis provides opportunities to better understand and manipulate the molecular biology of filariasis transmission.

Some strains of Bacillus sphaericus are entomopathogenic to mosquito larvae, which transmit diseases, such as filariasis and malaria, affecting millions of people worldwide. This species is unable to use hexoses and pentoses as unique carbon sources, which was proposed to be due to the lack of glycolytic enzymes, such as 6-phosphofructokinase (PFK). In this study, PFK activity was detected and the pfk gene was cloned and sequenced. Furthermore, this gene was shown to be present in strains belonging to all the homology groups of this heterogeneous species, in which PFK activity was also detected. A careful sequence analysis revealed the conservation of different catalytic and regulatory residues, as well as the enzyme's phylogenetic affiliation with the family of allosteric ATP-PFK enzymes.

Triatoma rubrovaria has become the most frequently captured triatomine species since the control of T. infestans in the State of Rio Grande do Sul (RS), Brazil. In order to evaluate the genetic variability of this species, field collections were performed in four municipalities where it has been reported and distant from 75 to 322 km. Specimens were analyzed by color pattern and isoenzymes. Nine enzymatic loci were interpreted from nine enzymatic systems. The Santiago population was isolated from the others with chromatic monomorphism and diagnostic alleles at Idh and Pgm loci. The study shows the existence of, at least, two distinct populations of T. rubrovaria in RS with different phenotypic and genetic pattern.