Comparative analysis of the complete genome of KPC-2-producing Klebsiella pneumoniae Kp13 reveals remarkable genome plasticity and a wide repertoire of virulence and resistance mechanisms.

BACKGROUND: Klebsiella pneumoniae is an important opportunistic pathogen associated with nosocomial and community-acquired infections. A wide repertoire of virulence and antimicrobial resistance genes is present in K. pneumoniae genomes, which can constitute extra challenges in the treatment of infections caused by some strains. K. pneumoniae Kp13 is a multidrug-resistant strain responsible for causing a large nosocomial outbreak in a teaching hospital located in Southern Brazil. Kp13 produces K. pneumoniae carbapenemase (KPC-2) but is unrelated to isolates belonging to ST 258 and ST 11, the main clusters associated with the worldwide dissemination of KPC-producing K. pneumoniae. In this report, we perform a genomic comparison between Kp13 and each of the following three K. pneumoniae genomes: MGH 78578, NTUH-K2044 and 342. RESULTS: We have completely determined the genome of K. pneumoniae Kp13, which comprises one chromosome (5.3 Mbp) and six plasmids (0.43 Mbp). Several virulence and resistance determinants were identified in strain Kp13. Specifically, we detected genes coding for six beta-lactamases (SHV-12, OXA-9, TEM-1, CTX-M-2, SHV-110 and KPC-2), eight adhesin-related gene clusters, including regions coding for types 1 (fim) and 3 (mrk) fimbrial adhesins. The rmtG plasmidial 16S rRNA methyltransferase gene was also detected, as well as efflux pumps belonging to five different families. Mutations upstream the OmpK35 porin-encoding gene were evidenced, possibly affecting its expression. SNPs analysis relative to the compared strains revealed 141 mutations falling within CDSs related to drug resistance which could also influence the Kp13 lifestyle. Finally, the genetic apparatus for synthesis of the yersiniabactin siderophore was identified within a plasticity region. Chromosomal architectural analysis allowed for the detection of 13 regions of difference in Kp13 relative to the compared strains. CONCLUSIONS: Our results indicate that the plasticity occurring at many hierarchical levels (from whole genomic segments to individual nucleotide bases) may play a role on the lifestyle of K. pneumoniae Kp13 and underlie the importance of whole-genome sequencing to study bacterial pathogens. The general chromosomal structure was somewhat conserved among the compared bacteria, and recombination events with consequent gain/loss of genomic segments appears to be driving the evolution of these strains.

Common ancestry of iron oxide- and iron-sulfide-based biomineralization in magnetotactic bacteria.

Magnetosomes are prokaryotic organelles produced by magnetotactic bacteria that consist of nanometer-sized magnetite (Fe(3)O(4)) or/and greigite (Fe(3)S(4)) magnetic crystals enveloped by a lipid bilayer membrane. In magnetite-producing magnetotactic bacteria, proteins present in the magnetosome membrane modulate biomineralization of the magnetite crystal. In these microorganisms, genes that encode for magnetosome membrane proteins as well as genes involved in the construction of the magnetite magnetosome chain, the mam and mms genes, are organized within a genomic island. However, partially because there are presently no greigite-producing magnetotactic bacteria in pure culture, little is known regarding the greigite biomineralization process in these organisms including whether similar genes are involved in the process. Here using culture-independent techniques, we now show that mam genes involved in the production of magnetite magnetosomes are also present in greigite-producing magnetotactic bacteria. This finding suggest that the biomineralization of magnetite and greigite did not have evolve independently (that is, magnetotaxis is polyphyletic) as once suggested. Instead, results presented here are consistent with a model in which the ability to biomineralize magnetosomes and the possession of the mam genes was acquired by bacteria from a common ancestor, that is, the magnetotactic trait is monophyletic.

Genomic and evolutionary comparisons of diazotrophic and pathogenic bacteria of the order Rhizobiales.

BACKGROUND: Species belonging to the Rhizobiales are intriguing and extensively researched for including both bacteria with the ability to fix nitrogen when in symbiosis with leguminous plants and pathogenic bacteria to animals and plants. Similarities between the strategies adopted by pathogenic and symbiotic Rhizobiales have been described, as well as high variability related to events of horizontal gene transfer. Although it is well known that chromosomal rearrangements, mutations and horizontal gene transfer influence the dynamics of bacterial genomes, in Rhizobiales, the scenario that determine pathogenic or symbiotic lifestyle are not clear and there are very few studies of comparative genomic between these classes of prokaryotic microorganisms trying to delineate the evolutionary characterization of symbiosis and pathogenesis. RESULTS: Non-symbiotic nitrogen-fixing bacteria and bacteria involved in bioremediation closer to symbionts and pathogens in study may assist in the origin and ancestry genes and the gene flow occurring in Rhizobiales. The genomic comparisons of 19 species of Rhizobiales, including nitrogen-fixing, bioremediators and pathogens resulted in 33 common clusters to biological nitrogen fixation and pathogenesis, 15 clusters exclusive to all nitrogen-fixing bacteria and bacteria involved in bioremediation, 13 clusters found in only some nitrogen-fixing and bioremediation bacteria, 01 cluster exclusive to some symbionts, and 01 cluster found only in some pathogens analyzed. In BBH performed to all strains studied, 77 common genes were obtained, 17 of which were related to biological nitrogen fixation and pathogenesis. Phylogenetic reconstructions for Fix, Nif, Nod, Vir, and Trb showed possible horizontal gene transfer events, grouping species of different phenotypes. CONCLUSIONS: The presence of symbiotic and virulence genes in both pathogens and symbionts does not seem to be the only determinant factor for lifestyle evolution in these microorganisms, although they may act in common stages of host infection. The phylogenetic analysis for many distinct operons involved in these processes emphasizes the relevance of horizontal gene transfer events in the symbiotic and pathogenic similarity.

Novel genes related to nodulation, secretion systems, and surface structures revealed by a genome draft of Rhizobium tropici strain PRF 81.

Rhizobium tropici is representative of the diversity of tropical rhizobia, besides comprising strains very effective in fixing N(2) in symbiosis with the common bean (Phaseolus vulgaris L.). The genome of a Brazilian commercial inoculant R. tropici strain (PRF 81, =SEMIA 4088), estimated at 7.85 Mb, was analyzed through a total of 9,026 shotgun reads, assembled in 1,668 phrap contigs, and covering approximately 30% of the genome. Annotation identified 2,135 coding DNA sequences (CDS), and only 57.2% have possible functions. The genome comprises a mosaic of genes, with CDS showing the highest similarities with 134 microorganisms, none of which represents more than 19% of the CDS with putative known functions. The high saprophytic capacity of PRF 81 may reside in a variety of genes related to transport, biodegradation of xenobiotics, defense, and secretion proteins, many of which were reported for the first time in the present study. Novelty was also found in nodulation (nodG, a double nodIJ system, nodT, nolF, nolG) and capsular polysaccharide genes, showing stronger similarities with Sinorhizobium (=Ensifer) than with the main symbionts of the common bean -- R. etli and R. leguminosarum -- suggesting that the original host of R. tropici might be another tropical legume or emphasizing the highly promiscuous nature of this rhizobial species.

Molecular phylogeny based on the 16S rRNA gene of elite rhizobial strains used in Brazilian commercial inoculants.

Nitrogen is often a limiting nutrient, therefore the sustainability of food crops, forages and green manure legumes is mainly associated with their ability to establish symbiotic associations with stem and root-nodulating N2-fixing rhizobia. The selection, identification and maintenance of elite strains for each host are critical. Decades of research in Brazil resulted in a list of strains officially recommended for several legumes, but their genetic diversity is poorly known. This study aimed at gaining a better understanding of phylogenetic relationships of 68 rhizobial strains recommended for 64 legumes, based on the sequencing of the 16S rRNA genes. The strains were isolated from a wide range of legumes, including all three subfamilies and 17 tribes. Nine main phylogenetic branches were defined, seven of them related to the rhizobial species: Bradyrhizobium japonicum, B. elkanii, Rhizobium tropici, R. leguminosarum, Sinorhizobium meliloti/S. fredii, Mesorhizobium ciceri/M. loti, and Azorhizobium caulinodans. However, some strains differed by up to 35 nucleotides from the type strains, which suggests that they may represent new species. Two other clusters included bacteria showing similarity with the genera Methylobacterium and Burkholderia, and amplification with primers for nifH and/or nodC regions was achieved with these strains. Host specificity of several strains was very low, as they were capable of nodulating legumes of different tribes and subfamilies. Furthermore, host specificity was not related to 16S rRNA, therefore evolution of ribosomal and symbiotic genes may have been diverse. Finally, the great diversity observed in this study emphasizes that tropics are an important reservoir of N2-fixation genes.