Structural organization of virulence-associated plasmids of Yersinia pestis

The complete nucleotide sequence and gene organization of the three virulence plasmids from Yersinia pestis KIM5 were determined. Plasmid pPCP1 (9,610 bp) has a GC content of 45.3% and encodes two previously known virulence factors, an associated protein, and a single copy of IS100. Plasmid pCD1 (70,504 bp) has a GC content of 44.8%. It is known to encode a number of essential virulence determinants, regulatory functions, and a multiprotein secretory system comprising the low-calcium response stimulation that is shared with the other two Yersinia species pathogenic for humans (Y. pseudotuberculosis and Y. enterocolitica). A new pseudogene, which occurs as an intact gene in the Y. enterocolitica and Y. pseudotuberculosis-derived analogues, was found in pCD1. It corresponds to that encoding the lipoprotein YlpA. Several intact and partial insertion sequences and/or transposons were also found in pCD1, as well as six putative structural genes with high homology to proteins of unknown function in other yersiniae. The sequences of the genes involved in the replication of pCD1 are highly homologous to those of the cognate plasmids in Y. pseudotuberculosis and Y. enterocolitica, but their localization within the plasmid differs markedly from those of the latter. Plasmid pMT1 (100,984 bp) has a GC content of 50.2%. It possesses two copies of IS100, which are located 25 kb apart and in opposite orientations. Adjacent to one of these IS100 inserts is a partial copy of IS285. A single copy of an IS200-like element (recently named IS1541) was also located in pMT1. In addition to 5 previously described genes, such as murine toxin, capsule antigen, capsule anchoring protein, etc., 30 homologues to genes of several bacterial species were found in this plasmid, and another 44 open reading frames without homology to any known or hypothetical protein in the databases were predicted.     

The genome of Staphylococcus aureus: a review

The genome of Staphylococcus aureus consists of a single circular chromosome (2.7-2.8 mbp) plus an assortment of extrachromosomal accessory genetic elements: conjugative and nonconjugative plasmids, mobile elements (IS, Tn, Hi), prophages and other variable elements. Plasmids (1-60 kbp) are classified into 4 classes and there are 15 known incompatibility groups. Mobile elements of the genome (0.8-18 kbp) appear in the chromosome or in plasmids of classes II and III. Prophages (45-60 kbp) are integrated in the bacterial chromosome, and they are UV- or mitomycin-inducible. Temperate bacteriophages of S. aureus are members of the Siphoviridae and the serological groups A, B and F occur most frequently. In the paper presented, the characteristics of chromosome, plasmids, transposons and other genetic elements of S. aureus genome are given and an alphabetical list of known genes of this species is included.     

Characterization of IS1547, a new member of the IS900 family in the Mycobacterium tuberculosis complex, and its association with IS6110

Unlike classically defined insertion sequence (IS) elements, which are delimited by their inverted terminal repeats, some IS elements do not have inverted terminal repeats. Among this group of atypical IS elements, IS116, IS900, IS901, and IS1110 have been proposed as members of the IS900 family of elements, not only because they do not have inverted terminal repeats but also because they share other features such as homologous transposases and particular insertion sites. In this study, we report a newly identified IS sequence, IS1547, which was first identified in a clinical isolate of Mycobacterium tuberculosis. Its structure, insertion site, and putative transposase all conform with the conventions of the IS900 family, suggesting that it is a new member of this family. IS1547 was detected only in isolates of the M. tuberculosis complex, where it had highly polymorphic restriction fragment length polymorphism patterns, suggesting that it may be a useful genetic marker for identifying isolates of the M. tuberculosis complex and for distinguishing different strains of M. tuberculosis. ipl is a preferential locus for IS6110 insertion where there are eight known different insertion sites for IS6110. Surprisingly, the DNA sequence of ipl is now known to be a part of IS1547, meaning that IS1547 is a preferential site for IS6110 insertion.     

Leptospira genomics

The bacterial species Leptospira interrogans (sensu stricto) has a complex genome containing two circular chromosomal replicons. Comparative analysis of the larger chromosome reveals a fluid genetic organization with many large rearrangements differentiating two closely related strains. In the present study new genes were identified by partial sequence analysis of randomly cloned fragments of L. interrogans DNA. These genes were localized in regions of the genome by nucleic acid hybridization with DNA fragments separated by pulsed-field gel electrophoresis. The resulting genetic maps provide improved resolution for each strain and provide evidence for additional chromosomal rearrangements. Insertion elements may be involved in recombination events, as several are near regions of the chromosome that have undergone rearrangement.     

Genome evolution within the alpha Proteobacteria: why do some bacteria not possess plasmids and others exhibit more than one different chromosome?

Animal intracellular Proteobacteria of the alpha subclass without plasmids and containing one or more chromosomes are phylogenetically entwined with opportunistic, plant-associated, chemoautotrophic and photosynthetic alpha Proteobacteria possessing one or more chromosomes and plasmids. Local variations in open environments, such as soil, water, manure, gut systems and the external surfaces of plants and animals, may have selected alpha Proteobacteria with extensive metabolic alternatives, broad genetic diversity, and more flexible and larger genomes with ability for horizontal gene flux. On the contrary, the constant and isolated animal cellular milieu selected heterotrophic alpha Proteobacteria with smaller genomes without plasmids and reduced genetic diversity as compared to their plant-associated and phototrophic relatives. The characteristics and genome sizes in the extant species suggest that a second chromosome could have evolved from megaplasmids which acquired housekeeping genes. Consequently, the genomes of the animal cell-associated Proteobacteria evolved through reductions of the larger genomes of chemoautotrophic ancestors and became rich in adenosine and thymidine, as compared to the genomes of their ancestors. Genome organisation and phylogenetic ancestor-descendent relationships between extant bacteria of closely related genera and within the same monophyletic genus and species suggest that some strains have undergone transition from two chromosomes to a single replicon. It is proposed that as long as the essential information is correctly expressed, the presence of one or more chromosomes within the same genus or species is the result of contingency. Genetic drift in clonal bacteria, such as animal cell-associated alpha Proteobacteria, would depend almost exclusively on mutation and internal genetic rearrangement processes. Alternatively, genomic variations in reticulate bacteria, such as many intestinal and plant cell-associated Proteobacteria, will depend not only on these processes, but also on their genetic interactions with other bacterial strains. Common pathogenic domains necessary for the invasion and survival in association with cells have been preserved in the chromosomes of the animal and plant-associated alpha Proteobacteria. These pathogenic domains have been maintained by vertical inherence, extensively ameliorated to match the chromosome G + C content and evolved within chromosomes of alpha Proteobacteria.     

"Black holes" and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli

Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylase (LDC) activity is present in approximately 90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these "black holes," deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.     

Skin grafting. From here to there

The transfer of genetic information between distantly or even unrelated organisms during evolution had been inferred from nucleotide sequence comparisons. These studies provided circumstantial evidence that in rare cases genes had been laterally transmitted amongst organisms of the domains bacteria, archaea and eukarya. Laboratory-based studies confirmed that the gene pools of the various domains of organisms are linked. Amongst the bacterial gene exchange mechanisms transduction, transformation and conjugation, the latter was identified as the mechanism with potentially the broadest host range of transfer. Previously, the issue of horizontal gene transfer has become important in the context of biosafety. Gene transfer studies carried out under more natural conditions such as in model ecosystems or in the environment established that all gene transfer mechanisms worked under these conditions. Moreover, environmental hot-spots were identified where favourable conditions such as nutrient enrichment increased the probability of genetic exchange among bacteria. In particular, the phytosphere was shown to provide conducive conditions for conjugative gene exchange. Concern has been expressed that transfer of recombinant DNA (e.g. antibiotic resistance genes) from genetically modified organisms (GMOs) such as transgenic plants to phytosphere bacteria may occur and thus contribute to the undesirable spread of antibiotic resistance determinants. Studies which were performed to address this issue clearly showed that such a transfer occurs, if at all, at extremely low frequency.     

Microbial toxins

Microbial toxins represent major virulence determinants of pathogenic bacteria. Endotoxins are lipopolysaccharides of the gram-negative bacterial cell wall. They exert their action via deregulating host cell function and stimulating cytokine overproduction. Exotoxins are proteins that can conveniently be grouped into three major categories. The first are intracellularly active molecules, all of which have to date been identified as enzymes. The second are membrane-damaging molecules, most of which create transmembrane pores. The third are the superantigens that cross-link T cells with antigen-presenting cells, thus causing cytokine overproduction. Most pathogens elaborate several toxins that can synergize in their detrimental action. Research on microbial toxins has fundamentally advanced our understanding of the pathogenesis of bacterial infections in the past two decades.     

The prevalence and characteristics of persistent pulmonary hypertension of the newborn. A multicenter study. The Study Group of the Società Italiana di Cardiologia Pediatrica (SICP)

Modifying bacterial virulence genes to probe the nature of host immunity is mostly unexplored. Here we investigate whether host immune responses can be regulated by modification of bacterial virulence genes. In mice, attenuated Salmonella mutant strains with clinical relevance elicited differential host immune responses. Oral administration of a mutant strain with a PhoP-null phenotype promoted potent innate immune responses of macrophages that were sufficient for host defense. In contrast, administration of an Aro- mutant strain elicited stronger specific antibody and T-helper (Th)-cell responses, wherein Th1-type cells were required for clearance. Thus, genetic manipulation of bacteria may be used to broadly alter immune mechanisms that regulate attenuation within the host and to tailor host immunity to specific bacterial pathogens.     

DNA sequencing and analysis of the low-Ca2+-response plasmid pCD1 of Yersinia pestis KIM5

The low-Ca2+-response (LCR) plasmid pCD1 of the plague agent Yersinia pestis KIM5 was sequenced and analyzed for its genetic structure. pCD1 (70,509 bp) has an IncFIIA-like replicon and a SopABC-like partition region. We have assigned 60 apparently intact open reading frames (ORFs) that are not contained within transposable elements. Of these, 47 are proven or possible members of the LCR, a major virulence property of human-pathogenic Yersinia spp., that had been identified previously in one or more of Y. pestis or the enteropathogenic yersiniae Yersinia enterocolitica and Yersinia pseudotuberculosis. Of these 47 LCR-related ORFs, 35 constitute a continuous LCR cluster. The other LCR-related ORFs are interspersed among three intact insertion sequence (IS) elements (IS100 and two new IS elements, IS1616 and IS1617) and numerous defective or partial transposable elements. Regional variations in percent GC content and among ORFs encoding effector proteins of the LCR are additional evidence of a complex history for this plasmid. Our analysis suggested the possible addition of a new Syc- and Yop-encoding operon to the LCR-related pCD1 genes and gave no support for the existence of YopL. YadA likely is not expressed, as was the case for Y. pestis EV76, and the gene for the lipoprotein YlpA found in Y. enterocolitica likely is a pseudogene in Y. pestis. The yopM gene is longer than previously thought (by a sequence encoding two leucine-rich repeats), the ORF upstream of ypkA-yopJ is discussed as a potential Syc gene, and a previously undescribed ORF downstream of yopE was identified as being potentially significant. Eight other ORFs not associated with IS elements were identified and deserve future investigation into their functions.     

Molecular changes in Entamoeba histolytica in response to bacteria

Entamoeba histolytica, the protozoan parasite, is the causative agent of amoebiasis. The degree of virulence, as inferred from invasiveness, of potentially pathogenic strains may be regulated by both host and parasite factors that determine the gut environment. One such factor that plays an important role is the bacterial flora in the gut. Previous studies have clearly shown that bacterial flora is an important determinant of virulence in E. histolytica. However, the exact nature of changes induced in E. histolytica in response to bacteria and their role in virulence is not clear. In this study the levels of a number of molecules potentially important in virulence mechanisms were determined in E. histolytica cells grown with and without normal human bacterial flora, using enzyme-linked immunosorbent assay. Significant changes were observed only after the E. histolytica cells had been adapted to grow with bacterial flora for a number of generations, and not in short term culture.     

At least four percent of the Salmonella typhimurium genome is required for fatal infection of mice

Salmonella typhimurium infection of mice is an established model system for studying typhoid fever in humans. Using this model, we identified S. typhimurium genes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo. Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1, 000 times that of the parental strain were identified. These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions. In addition, the corresponding transposon insertions were mapped within the S. typhimurium genome and the nucleotide sequence of the transposon-flanking DNA was obtained. Salmonella spp. and related bacteria were probed with flanking DNA for the presence of these genes. All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of these were previously described. Furthermore, the proportion of attenuated mutants obtained in this study suggests that mutations in about 4% of the Salmonella genome lead to 1,000-fold or greater attenuation in the mouse typhoid model of infection. Most of these genes appear to be required during the early stages of a natural infection.     

Pathogenic power of Salmonellae: virulence factors and study models

Salmonellae are potentially pathogenic for humans as well as for numerous animal species. They possess numerous virulence factors, which allow them to adapt to various environmental conditions and to host response at each step of the pathogenic process. Key-steps such as the invasion of epithelial cells or survival within macrophages have been extensively studied. These studies have led to the discovery of an original protein secretion system and have demonstrated the existence of pathogenicity islands. This considerable progress is due to the development of numerous in vitro and in vivo models and of new identification strategies for the implicated genes. Recently, many original and elegant strategies have been recently proposed.     

katGI and katGII encode two different catalases-peroxidases in Mycobacterium fortuitum

It has been suggested that catalase-peroxidase plays an important role in several aspects of mycobacterial metabolism and is a virulence factor in the main pathogenic mycobacteria. In this investigation, we studied genes encoding for this protein in the fast-growing opportunistic pathogen Mycobacterium fortuitum. Nucleotide sequences of two different catalase-peroxidase genes (katGI and katGII) of M. fortuitum are described. They show only 64% homology at the nucleotide level and 55% identity at the amino acid level, and they are more similar to catalases-peroxidases from different bacteria, including mycobacteria, than to each other. Both proteins were found to be expressed in actively growing M. fortuitum, and both could also be expressed when transformed into Escherichia coli and M. aurum. We detected the presence of a copy of IS6100 in the neighboring region of a katG gene in the M. fortuitum strain in which this element was identified (strain FC1). The influence of each katG gene on isoniazid (isonicotinic acid hydrazide; INH) susceptibility of mycobacteria was checked by using the INH-sensitive M. aurum as the host. Resistance to INH was induced when katGI was transformed into INH-sensitive M. aurum, suggesting that this enzyme contributes to the natural resistance of M. fortuitum to the drug. This is the first report showing two different genes encoding same enzyme activity which are actively expressed within the same mycobacterial strain.     

Virulence mechanisms of avian fimbriated Escherichia coli in experimentally inoculated chickens

Virulence mechanisms of avian pathogenic Escherichia coli were investigated by inoculating commercial broiler chickens via the left caudal thoracic air sac with three highly pathogenic and three less pathogenic E. coli isolates. At 6 h postinoculation, all isolates had colonized the respiratory tract (trachea, lungs, and air sacs) and internal organs (liver, spleen, and kidney) of inoculated birds, but bacteria were recovered from pericardial fluid and blood only of birds inoculated with the more pathogenic isolates. F1 fimbriae were expressed on a high proportion of bacteria colonizing the trachea and to a lesser extent on bacteria in the lungs of birds inoculated with each of the isolates. F1 fimbriae were also expressed on bacteria in air sacs only for the less pathogenic isolates. P(F11) fimbriae were expressed on bacteria present in air sacs, lungs, kidney, blood, and pericardial fluid of birds inoculated with one of the more virulent isolates. On electron microscopy, bacteria of the more pathogenic isolates but not of the less pathogenic isolates were observed often associated with or within macrophages, which appeared to be viable, in the air sacs and lungs. In in vitro assays, the more pathogenic but not the less pathogenic isolates, were resistant to opsonization and phagocytosis in the absence of F1 fimbriae, whereas bacteria of all isolates were rapidly killed by avian macrophages when they expressed F1 fimbriae. These results suggest that resistance to phagocytosis may be an important mechanism in avian colisepticemia. They also suggest that F1 fimbrial phase variation to the nonfimbriated phase is favored in the avian lower respiratory tract, is more marked for the more pathogenic-isolates, and may be a virulence mechanism.     

Analyzing genomes with cumulative skew diagrams

A novel method of cumulative diagrams shows that the nucleotide composition of a microbial chromosome changes at two points separated by about a half of its length. These points coincide with sites of replication origin and terminus for all bacteria where such sites are known. The leading strand is found to contain more guanine than cytosine residues. This fact is used to predict origin and terminus locations in other bacterial and archaeal genomes. Local changes, visible as diagram distortions, may represent recent genome rearrangements, as demonstrated for two strains of Escherichia coli . Analysis of the diagrams of viral and mitochondrial genomes suggests a link between the base composition bias and the time spent by DNA in a single stranded state during replication.