Gene expression of Vibrio parahaemolyticus growing in laboratory isolation conditions compared to those common in its natural ocean environment.

BACKGROUND: Vibrio parahaemolyticus is an autochthonous marine bacterial species comprising strains able to grow in broth containing bile salts at 37 °C, a condition seldom found in the ocean. However, this condition is used for isolation in the laboratory because it is considered a necessary property for pathogenesis. In this context, revealing how gene expression enables V. parahaemolyticus to adapt to this particular condition -common to almost all V. parahaemolyticus isolates- will improve our understanding of the biology of this important pathogen. To determine the genes of V. parahaemolyticus differentially expressed when growing in isolation condition (37 °C, 0.9% NaCl, and 0.04% bile salts) referred to those at the temperature and salt concentration prevailing in ocean south of Chile (marine-like condition; 12 °C, 3% NaCl, and absence of bile salts) we used high-throughput sequencing of RNA. RESULTS: Our results showed that in the isolation condition, among the 5034 genes annotated in the V. parahaemolyticus RIMD2210633 genome, 344 were upregulated and 433 downregulated referred to the marine-like condition, managing an adjusted P-value (Padj) < E-5. Between the 50 more highly expressed genes, among the small RNAs (sRNA), the three carbon storage regulators B (CsrB) were up four to six times, while RyhB, related to iron metabolism besides motility control, was down about eight times. Among proteins, BfdA, a hemolysin-co-regulated protein (Hcp1) secreted by T6SS1, one of the most highly expressed genes, was about 140 times downregulated in isolation condition. The highest changes in relative expression were found among neighboring genes coding for proteins related to respiration, which were about 40 times upregulated. CONCLUSIONS: When V. parahaemolyticus is grown in conditions used for laboratory isolation 777 genes are up- or downregulated referred to conditions prevailing in the marine-like condition; the most significantly overrepresented categories among upregulated processes were those related to transport and localization, while secretion and pathogenesis were overrepresented among downregulated genes. Genes with the highest differential expression included the sRNAs CsrB and RhyB and the mRNAs related with secretion, nutritional upshift, respiration and rapid growing.

Genome diversification within a clonal population of pandemic Vibrio parahaemolyticus seems to depend on the life circumstances of each individual bacteria.

BACKGROUND: New strains of Vibrio parahaemolyticus that cause diarrhea in humans by seafood ingestion periodically emerge through continuous evolution in the ocean. Influx and expansion in the Southern Chilean ocean of a highly clonal V. parahaemolyticus (serotype O3:K6) population from South East Asia caused one of the largest seafood-related diarrhea outbreaks in the world. Here, genomics analyses of isolates from this rapidly expanding clonal population offered an opportunity to observe the molecular evolutionary changes often obscured in more diverse populations. RESULTS: Whole genome sequence comparison of eight independent isolates of this population from mussels or clinical cases (from different years) was performed. Differences of 1366 to 217,729 bp genome length and 13 to 164 bp single nucleotide variants (SNVs) were found. Most genomic differences corresponded to the presence of regions unique to only one or two isolates, and were probably acquired by horizontal gene transfer (HGT). Some DNA gain was chromosomal but most was in plasmids. One isolate had a large region (8,644 bp) missing, which was probably caused by excision of a prophage. Genome innovation by the presence of unique DNA, attributable to HGT from related bacteria, varied greatly among the isolates, with values of 1,366 (ten times the number of highest number of SNVs) to 217,729 (a thousand times more than the number of highest number of SNVs). CONCLUSIONS: The evolutionary forces (SNVs, HGT) acting on each isolate of the same population were found to differ to an extent that probably depended on the ecological scenario and life circumstances of each bacterium.

Rise and fall of pandemic Vibrio parahaemolyticus serotype O3:K6 in southern Chile.

Seafood consumption-related diarrhoea increased drastically in Chile when the pandemic strain of Vibrio parahaemolyticus serotype O3:K6 reached Region de Los Lagos, where most of Chile's seafood is produced. Outbreaks peaked in 2005 with 3725 clinical cases in this region and gradually decreased to fewer than 10 cases in 2010 and 2011. We show here that the pandemic strain concurrently vanished from mussels; we also report further environmental data. Integration of the 2010/2011 data with those obtained since 2004 suggests that after its arrival in southern Chile, the pandemic strain grew in mussels, likely facilitated by a minor rise in surface seawater temperature and by warming of the mussels in the intertidal region due to frequent sunny days. However, since these environmental parameters probably equally affected the pandemic strain and more than 30 V. parahaemolyticus DNA restriction clusters that inhabit local shellfish, a selective effect of bacteriophages is proposed. Lytic bacteriophage VP93 may have favoured the growth of the pandemic strain versus similar phage-sensitive strains, as shown here in a particular case. However, the pandemic strain's decline may have been promoted by temperate phage VP58.5, which kills the pandemic strain and increases the UV sensitivity of lysogenized phage-resistant cells.

A new group of cosmopolitan bacteriophages induce a carrier state in the pandemic strain of Vibrio parahaemolyticus.

A clonal population of pathogenic Vibrio parahaemolyticus O3 : K6 serovar has spread in coastal waters, causing outbreaks worldwide since 1996. Bacteriophage infection is one of the main factors affecting bacterial strain concentration in the ocean. We studied the occurrence and properties of phages infecting this V. parahaemolyticus pandemic strain in coastal waters. Analysing 143 samples, phages were found in 13. All isolates clustered in a closely related group of podophages with at least 90% nucleotide sequence identity in three essential genes, despite distant geographical origins. These bacteriophages were able to multiply on the V. parahaemolyticus pandemic strain, but the impact on host concentration and subsequent growth was negligible. Infected bacteria continued producing the phage but were not lysogenized. The phage genome of prototype strain VP93 is 43 931 nucleotides and contains 337 bp direct terminal repeats at both ends. VP93 is the first non-Pseudomonas phage related to the PhiKMV-like subgroup of the T7 supergroup. The lack of a major effect on host growth suggests that these phages exert little control on the propagation of the pandemic strain in the environment. This form of phage growth can be modelled if phage-sensitive and -resistant cells that convert to each other with a high frequency are present in clonal cultures of pandemic V. parahaemolyticus.

The linear plasmid prophage Vp58.5 of Vibrio parahaemolyticus is closely related to the integrating phage VHML and constitutes a new incompatibility group of telomere phages.

Vibrio parahaemolyticus O3:K6 pandemic strains recovered in Chile frequently possess a 42-kb plasmid which is the prophage of a myovirus. We studied the prototype phage VP58.5 and show that it does not integrate into the host cell chromosome but replicates as a linear plasmid (Vp58.5) with covalently closed ends (telomeres). The Vp58.5 replicon coexists with other plasmid prophages (N15, PY54, and PhiKO2) in the same cell and thus belongs to a new incompatibility group of telomere phages. We determined the complete nucleotide sequence (42,612 nucleotides) of the VP58.5 phage DNA and compared it with that of the plasmid prophage. The two molecules share the same nucleotide sequence but are 35% circularly permuted to each other. In contrast to the hairpin ends of the plasmid, VP58.5 phage DNA contains 5'-protruding ends. The VP58.5 sequence is 92% identical to the sequence of phage VHML, which was reported to integrate into the host chromosome. However, the gene order and termini of the phage DNAs are different. The VHML genome exhibits the same gene order as does the Vp58.5 plasmid. VHML phage DNA has been reported to contain terminal inverted repeats. This repetitive sequence is similar to the telomere resolution site (telRL) of VP58.5 which, after processing by the phage protelomerase, forms the hairpin ends of the Vp58.5 prophage. It is discussed why these closely related phages may be so different in terms of their genome ends and their lifestyle.

Origins and colonization history of pandemic Vibrio parahaemolyticus in South America.

The dynamics of dissemination of the environmental human pathogen Vibrio parahaemolyticus are uncertain. The O3:K6 clone was restricted to Asia until its detection along the Peruvian coasts and in northern Chile in 1997 in phase with the arrival of El Niño waters. A subsequent emergence of O3:K6 strains was detected in austral Chile in 2004. The origin of these 1997 and 2004 population radiations has not yet been conclusively determined. Multiple loci VNTR analysis using seven polymorphic loci was carried out with a number of representative strains from Asia, Peru and Chile to determine their genetic characteristics and population structure. Asian and Chilean subpopulations were the most genetically distant groups with an intermediate subpopulation in Peru. Population structure inferred from a minimum-spanning tree and Bayesian analysis divided the populations into two genetically distinct groups, consistent with the epidemic dynamics of the O3:K6 clone in South America. One group comprised strains from the original Asiatic population and strains arriving in Peru and Chile in 1997. The second group included the remaining Peruvian Strains and Chilean strains obtained from Puerto Montt in 2004. The analysis of the arrival of the O3:K6 clone at the Pacific coasts of South America has provided novel insights linking the origin of the invasion in 1997 to Asian populations and describing the successful establishment of the O3:K6 populations, first in Peru and subsequently in the South of Chile owing to a possible radiation of Peruvian populations.

Epidemiology of Vibrio parahaemolyticus outbreaks, southern Chile.

Disease outbreaks caused by Vibrio parahaemolyticus in Puerto Montt, Chile, began in 2004 and reached a peak in 2005 at 3,600 clinical cases. Until 2006, every analyzed case was caused by the serovar O3:K6 pandemic strain. In the summer of 2007, only 475 cases were reported; 73% corresponded to the pandemic strain. This decrease was associated with a change in serotype of many pandemic isolates to O3:K59 and the emergence of new clinical strains. One of these strains, associated with 11% of the cases, was genotypically different from the pandemic strain but contained genes that were identical to those found on its pathogenicity island. These findings suggest that pathogenicity-related genes were laterally transferred from the pandemic strain to one of the different V. parahaemolyticus groups comprising the diverse and shifting bacterial population in shellfish in this region.

Enhancement of UV light sensitivity of a Vibrio parahaemolyticus O3:K6 pandemic strain due to natural lysogenization by a telomeric phage.

The Vibrio parahaemolyticus O3:K6 pandemic clonal strain was first observed in southern Chile in 2004 and has since caused approximately 8,000 seafood-related diarrhea cases in this region. The massive proliferation of the original clonal population offers a unique opportunity to study the evolution of a bacterial pathogen in its natural environment by detection and characterization of emerging bacterial variants. Here, we describe a group of pandemic variants characterized by the presence of a 42-kb extrachromosomal DNA that can be recovered by alkaline extraction. Upon treatment with mitomycin C, these variants lyse with production of a myovirus containing DNA of equal size to the plasmid but which cannot be recovered by alkaline extraction. Plasmid and phage DNAs show similar restriction patterns corresponding to enzyme sites in a circular permutation. Sequenced regions showed 81 to 99% nucleotide similarity to bacteriophage VHML of Vibrio harveyi. Altogether these observations indicate that the 42-kb plasmid corresponds to a prophage, consisting of a linear DNA with terminal hairpins of a telomeric temperate phage with a linear genome. Bacteria containing the prophage were 7 to 15 times more sensitive to UV radiation, likely due to phage induction by UV irradiation as plasmid curing restored the original sensitivity. The enhanced UV sensitivity could have a significant role in reducing the survival and propagation capability of the V. parahaemolyticus pandemic strain in the ocean.

Multiple-locus variable-number tandem-repeat analysis for clonal identification of Vibrio parahaemolyticus isolates by using capillary electrophoresis.

Epidemics of Vibrio parahaemolyticus in Chile have occurred since 1998. Direct genome restriction enzyme analysis (DGREA) using conventional gel electrophoresis permitted discrimination of different V. parahaemolyticus isolates obtained from these outbreaks and showed that this species consists of a highly diverse population. A multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) approach was developed and applied to 22 clinical and 91 environmental V. parahaemolyticus isolates from Chile to understand their clonal structures. To this end, an advanced molecular technique was developed by applying multiplex PCR, fluorescent primers, and capillary electrophoresis, resulting in a high-resolution and high-throughput (HRHT) genotyping method. The genomic basis of this HRHT method was eight VNTR loci described previously by Kimura et al. (J. Microbiol. Methods 72:313-320, 2008) and two new loci which were identified by a detailed molecular study of 24 potential VNTR loci on both chromosomes. The isolates of V. parahaemolyticus belonging to the same DGREA pattern were distinguishable by the size variations in the indicative 10 VNTRs. This assay showed that these 10 VNTR loci were useful for distinguishing isolates of V. parahaemolyticus that had different DGREA patterns and also isolates that belong to the same group. Isolates that differed in their DGREA patterns showed polymorphism in their VNTR profiles. A total of 81 isolates was associated with 59 MLVA groups, providing fine-scale differentiation, even among very closely related isolates. The developed approach enables rapid and high-resolution analysis of V. parahaemolyticus with pandemic potential and provides a new surveillance tool for food-borne pathogens.

Dynamics of clinical and environmental Vibrio parahaemolyticus strains during seafood-related summer diarrhea outbreaks in southern Chile.

Seafood consumption-related diarrhea became prevalent in Chile when the pandemic strain of Vibrio parahaemolyticus serotype O3:K6 reached a region in the south of Chile (Region de los Lagos) where approximately 80% of the country's seafood is produced. In spite of the large outbreaks of clinical infection, the load of V. parahaemolyticus in shellfish of this region is relatively low. The pandemic strain constitutes a small but relatively stable group of a diverse V. parahaemolyticus population, composed of at least 28 genetic groups. Outbreaks in Region de los Lagos began in 2004 and reached a peak in 2005 with 3,725 clinical cases, all associated with the pandemic strain. After 2005, reported cases steadily decreased to a total of 477 cases in 2007. At that time, 40% of the clinical cases were associated with a pandemic strain of a different serotype (O3:K59), and 27% were related to V. parahaemolyticus isolates unrelated to the pandemic strain. In the results published here, we report that in the summer of 2008, when reported cases unexpectedly increased from 477 to 1,143, 98% of the clinical cases were associated with the pandemic strain serotype O3:K6, a change from 2007. Nevertheless, in 2009, when clinical cases decreased to 441, only 64% were related to the pandemic strain; the remaining cases were related to a nonpandemic tdh- and trh-negative strain first identified in shellfish in 2006. Overall, our observations indicate that the pandemic strain has become a relatively stable subpopulation and that when the number of diarrhea cases related to the pandemic strain is low, previously undetected V. parahaemolyticus pathogenic strains become evident.

Determination of molecular phylogenetics of Vibrio parahaemolyticus strains by multilocus sequence typing.

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. There is a growing public health concern due to the emergence of a pandemic strain causing severe outbreaks worldwide. Many questions remain unanswered regarding the evolution and population structure of V. parahaemolyticus. In this work, we describe a multilocus sequence typing (MLST) scheme for V. parahaemolyticus based on the internal fragment sequences of seven housekeeping genes. This MLST scheme was applied to 100 V. parahaemolyticus strains isolated from geographically diverse clinical (n = 37) and environmental (n = 63) sources. The sequences obtained from this work were deposited and are available in a public database (http://pubmlst.org/vparahaemolyticus). Sixty-two unique sequence types were identified, and most (50) were represented by a single isolate, suggesting a high level of genetic diversity. Three major clonal complexes were identified by eBURST analysis. Separate clonal complexes were observed for V. parahaemolyticus isolates originating from the Pacific and Gulf coasts of the United States, while a third clonal complex consisted of strains belonging to the pandemic clonal complex with worldwide distribution. The data reported in this study indicate that V. parahaemolyticus is genetically diverse with a semiclonal population structure and an epidemic structure similar to that of Vibrio cholerae. Genetic diversity in V. parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation, with recombination ratios estimated at 2.5:1 and 8.8:1 by allele and site, respectively. Application of this MLST scheme to more V. parahaemolyticus strains and by different laboratories will facilitate production of a global picture of the epidemiology and evolution of this pathogen.

Multiple Ribosomal RNA Operons in Bacteria; Their Concerted Evolution and Potential Consequences on the Rate of Evolution of Their 16S rRNA.

Bacterial species differ greatly in the number and location of the rRNA operons which may be present in the bacterial chromosomes and plasmids. Most bacterial species contain more than one ribosomal RNA operon copy in their genomes, with some species containing up to 15 such copies. We review the number and location of the rRNA operons and discuss evolution of 16S rRNA (rrs) genes -which are considered as ultimate chronometers for phylogenetic classification- in bacteria with multiple copies of these genes. In these bacterial species, the rrs genes must evolve in concert and sequence changes generated by mutation or horizontal gene transfer must be either erased or spread to every gene copy to avoid divergence, as it occurs when they are present in different species. Analysis of polymorphic sites in intra-genomic rrs copies identifies putative conversion events and demonstrates that sequence conversion is patchy and occurs in small conversion tracts. Sequence conversion probably arises by a non-reciprocal transfer between two or more copies where one copy contributes only a small contiguous segment of DNA, whereas the other copy contributes the rest of the genome in a fairly well understood molecular process. Because concerted evolution implies that a mutation in any of the rrs copies is either eliminated or transferred to every rrs gene in the genome, this process should slow their evolution rate relative to that of single copy genes. However, available data on the rrs genes in bacterial genomes do not show a clear relationship between their evolution rates and the number of their copies in the genome.

Vibrio parahaemolyticus strains isolated during investigation of the summer 2006 seafood related diarrhea outbreaks in two regions of Chile.

Nine hundred cases of seafood related diarrhea were reported in the region of Puerto Montt, Chile during the austral summer of 2006. This is the continuation of the large outbreaks associated with the consumption of seafood containing the Vibrio parahaemolyticus serovar O3:K6 pandemic clonal group that arose last decade in Chile. The initial outbreaks occurred during the summer of 1998 in Antofagasta (23 degrees 39'S 70 degrees 24'W). Subsequently, outbreaks there were rare, but since 2004 outbreaks have been frequent farther south in Puerto Montt (41 degrees 29'S 72 degrees 24'W). The large outbreaks in Puerto Montt and their rarity in Antofagasta is atypical because the seawater temperature at Puerto Montt is 5 degrees C lower than at Antofagasta and the presence of V. parahaemolyticus in seafood has been associated with higher water temperatures. To better understand the role of seafood in outbreak occurrences in these regions, we analyzed the V. parahaemolyticus populations in clinical cases and shellfish from Puerto Montt during diarrhea outbreaks in 2006 and in shellfish from Antofagasta, where no cases were observed. Enrichment culture from shellfish yielded no V. parahaemolyticus from samples from the north, but its presence was detected in 80% of the samples from the south. Grouping of the V. parahaemolyticus isolates by the fragment restriction pattern of their DNA showed that all pathogenic (tdh+) isolates obtained from Puerto Montt shellfish corresponded to the serovar O3:K6 South East Asian pandemic clone, while the non-pathogenic (tdh-) isolates corresponded to at least six discrete groups. The possible causes for the disappearance of the pandemic strain from the north and its persistence in the south are discussed.

Insight Into the Origin and Evolution of the Vibrio parahaemolyticus Pandemic Strain.

A strain of Vibrio parahaemolyticus that emerged in 1995 caused the first known pandemic involving this species. This strain comprises clonal autochthonous ocean-dwelling bacteria whose evolution has occurred in the ocean environment. The low sequence diversity in this population enabled the discovery of information on its origin and evolution that has been hidden in bacterial clones that have evolved over a long period. Multilocus sequencing and microarray analysis, together with phylogenetic analysis, of pandemic and pre-pandemic isolates has suggested that the founder clone was an O3:K6 non-pathogenic strain that initially acquired a toxRS/new region and subsequently acquired at least seven novel genomic islands. Sequencing and comparison of whole genomes later confirmed these early observations, and it confirmed that most of the genetic changes occurred via gene conversion involving horizontally transmitted DNA. The highly clonal population rapidly diversified, especially in terms of antigenicity, and 27 serotypes have already been reported. Comparisons of the core genomes derived from the founder clone indicate that there are only a few hundred single-nucleotide variations between isolates. However, when the whole genome is considered (the core plus non-core genome and from any clonal frame), the amount of DNA with a different clonal frame can reach up to 4.2% and the number of single-nucleotide variations can reach several hundred thousand. Altogether, these and previous observations based on multilocus sequence typing, microarray analysis, and whole-genome sequencing indicate the large contribution made by DNA with different clonal genealogy to genome diversification. The evidence also indicates that horizontal gene transfer (HGT) caused the emergence of new pathogens. Furthermore, the extent of HGT seems to depend on the vicissitudes of the life of each bacterium, as exemplified by differences in thousands of base pairs acquired by HGT among almost identical genetic isolates.

Variation in the 16S-23S rRNA intergenic spacer regions in Vibrio parahaemolyticus strains are due to indels nearby their tRNAGlu.

Vibrio parahaemolyticus contains 11 rRNA operons each including one of six 16S-23S rRNA gene intergenic spacer classes differing in size and nucleotide sequence. Some of the spacer classes may differ between isolates. We observed that the differences in the spacers between isolates are generally in two spacer classes present in single copies in the genome, one class containing tRNA(Ala) and tRNA(Glu) and the other tRNA(Glu) exclusively. Moreover, these differences are due to indels located nearby their tRNA(Glu). Comparison of the nucleotide sequence between spacer classes suggests that intragenomic nonreciprocal recombination causes the size variations observed in the spacer regions of V. parahaemolyticus strains.

Role of Non-coding Regulatory RNA in the Virulence of Human Pathogenic Vibrios.

In recent decades, the identification of small non-coding RNAs in bacteria has revealed an important regulatory mechanism of gene expression involved in the response to environmental signals and to the control of virulence. In the family Vibrionaceae, which includes several human and animal pathogens, small non-coding RNAs (sRNAs) are closely related to important processes including metabolism, quorum sensing, virulence, and fitness. Studies conducted in silico and experiments using microarrays and high-throughput RNA sequencing have led to the discovery of an unexpected number of sRNAs in Vibrios. The present review discusses the most relevant reports regarding the mechanisms of action of sRNAs and their implications in the virulence of the main human pathogens in the family Vibrionaceae: Vibrio parahaemolyticus, V. vulnificus and V. cholerae.

Polymorphism and gene conversion of the 16S rRNA genes in the multiple rRNA operons of Vibrio parahaemolyticus.

The genome sequence of a strain of Vibrio parahaemolyticus holds 11 copies of rRNA operons (rrn) with identical 16S rRNA genes (rrs). Conversely, the species type strain contains two rrs classes differing in 10 nucleotide sites within a short segment of 25 bp. Furthermore, we show here that the sequence of this particular segment largely differs between some strains of this species. We also show that of the eleven rrn operons in the species type strain, seven contain one rrs class and four the other, indicating gene conversion. Our results support the hypothesis that the rrs differences observed between strains of this species were caused by lateral transfer of an rrs segment and subsequent conversion.

Inactivated E. coli transformed with plasmids that produce dsRNA against infectious salmon anemia virus hemagglutinin show antiviral activity when added to infected ASK cells.

Infectious salmon anemia virus (ISAV) has caused great losses to the Chilean salmon industry, and the success of prevention and treatment strategies is uncertain. The use of RNA interference (RNAi) is a promising approach because during the replication cycle, the ISAV genome must be transcribed to mRNA in the cytoplasm. We explored the capacity of E. coli transformed with plasmids that produce double-stranded RNA (dsRNA) to induce antiviral activity when added to infected ASK cells. We transformed the non-pathogenic Escherichia coli HT115 (DE3) with plasmids that expressed highly conserved regions of the ISAV genes encoding the nucleoprotein (NP), fusion (F), hemagglutinin (HE), and matrix (M) proteins as dsRNA, which is the precursor of the RNAi mechanism. The inactivated transformed bacteria carrying dsRNA were tested for their capacity to silence the target ISAV genes, and the dsRNA that were able to inhibit gene expression were subsequently tested for their ability to attenuate the cytopathic effect (CPE) and reduce the viral load. Of the four target genes tested, inactivated E. coli transformed with plasmids producing dsRNA targeting HE showed antiviral activity when added to infected ASK cells.

Characterization and identification of an iron-oxidizing, Leptospirillum-like bacterium, present in the high sulfate leaching solution of a commercial bioleaching plant.

Most copper bioleaching plants operate with a high concentration of sulfate salts, caused by the continuous addition of sulfuric acid and the recycling of the leaching solution. Since the bacteria involved in bioleaching have been generally isolated at low sulfate concentrations, the bacterial population present in the high-sulfate (150 gl(-1)) leaching solution, employed in a copper production plant, was investigated. The iron-oxidizing bacteria able to grow in the leaching solution were enriched by several batch cultivations and, after serial dilution, an abundant bacterial strain was isolated. This strain, called LA, exhibited a relatively constant rate of iron-oxidation in media containing sulfate ions at concentrations ranging from 10 to 150 gl(-1). Culture collection strains of Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans showed limited abilities to grow at sulfate ion concentrations higher than 70 gl(-1). In spite of its tolerance to high sulfate concentrations, strain LA was as sensitive to NaCl as A. ferrooxidans. Comparative sequence analysis of the 16S rRNA gene of strain LA indicated that it is phylogenetically related to strains described as Leptospirillum ferrooxidans. Bacterial community DNA restriction patterns of 16S rRNA genes suggested that strain LA was a minor component of the bacterial population present in leaching solution, but is abundant in ore leached with this solution.

Comprehensive detection of bacterial populations by PCR amplification of the 16S-23S rRNA spacer region.

PCR amplification of the spacer region between the 16S and 23S rRNA genes is commonly employed for the analysis of bacterial communities. In this analysis, the intergenic spacers are amplified by PCR using primers complementary to conserved regions in the 3' 16S rDNA and 5' 23S rDNA. By this method, the observation of every bacterial population may be limited by several causes. To explore the extent of bacterial populations overlooked by this method, we have used an empirical approach. In a sample containing about 50 colonies, we tested the capability to amplify by PCR the spacers from each colony. We also examined the ability to observe the spacers from each colony in the product obtained after amplification of the DNA extracted from the whole sample, as it is usually performed by this method. Contrarily to our expectations that a significant fraction of colonies would not yield amplification products, spacers were successfully amplified from every colony of two different samples examined. Overall, our results suggest that in spite of well-based theoretical limitations, the analysis of bacterial communities by amplification of the spacer regions can render a comprehensive representation of the more abundant bacterial clades in the sample.