Comparative analysis of the diversity of trinucleotide repeats in bacterial genomes.

The human gut is the most favorable niche for microbial populations, and few studies have explored the possibilities of horizontal gene transfer between host and pathogen. Trinucleotide repeat (TNR) expansion in humans can cause more than 40 neurodegenerative diseases. Further, TNRs are a type of microsatellite that resides on coding regions can contribute to the synthesis of homopolymeric amino acids. Hence, the present study aims to estimate the occurrence and diversity of TNRs in bacterial genomes available in the NCBI Genome database. Genome-wide analyses revealed that several bacterial genomes contain different types of uninterrupted TNRs. It was found that TNRs are abundant in the genomes of Alcaligenes faecalis, Mycoplasma gallisepticum, Mycoplasma genitalium, Sorangium cellulosum, and Thermus thermophilus. Interestingly, the genome of Bacillus thuringiensis strain YBT-1518 contained 169 uninterrupted ATT repeats. The genome of Leclercia adecarboxylata had 46 uninterrupted CAG repeats, which potentially translate into polyglutamine. In some instances, the TNRs were present in genes that potentially encode essential functions. Similar occurrences in human genes are known to cause genetic disorders. Further analysis of the occurrence of TNRs in bacterial genomes is likely to provide a better understanding of mismatch repair, genetic disorders, host-pathogen interaction, and homopolymeric amino acids.

Comparative genome analysis of Pasteurella multocida strains of porcine origin.

Pasteurella multocida causes acute/chronic pasteurellosis in porcine, resulting in considerable economic losses globally. The draft genomes of two Indian strains NIVEDIPm17 (serogroup D) and NIVEDIPm36 (serogroup A) were sequenced. A total of 2182-2284 coding sequences (CDSs) were predicted along with 5-6 rRNA and 45-46 tRNA genes in the genomes. Multilocus sequence analysis and LPS genotyping showed the presence of ST50: genotype 07 and ST74: genotype 06 in NIVEDIPm17 and NIVEDIPm36, respectively. Pangenome analysis of 61 strains showed the presence of 1653 core genes, 167 soft core genes, 750 shell genes, and 1820 cloud genes. Analysis of virulence-associated genes in 61 genomes indicated the presence of nanB, exbB, exbD, ptfA, ompA, ompH, fur, plpB, fimA, sodA, sodC, tonB, and omp87 in all strains. The 61 genomes contained genes encoding tetracycline (54%), streptomycin (48%), sulphonamide (28%), tigecycline (25%), chloramphenicol (21%), amikacin (7%), cephalosporin (5%), and trimethoprim (5%) resistance. Multilocus sequence type revealed that ST50 was the most common (34%), followed by ST74 (26%), ST13 (24%), ST287 (5%), ST09 (5%), ST122 (3%), and ST07 (2%). Single-nucleotide polymorphism and core genome-based phylogenetic analysis clustered the strains into three major clusters. In conclusion, we described the various virulence factors, mobile genetic elements, and antimicrobial resistance genes in the pangenome of P. multocida of porcine origin, besides the rare presence of LPS genotype 7 in serogroup D.

Comparative genomics of the Pasteurella multocida toxin.

Pasteurella multocida is a zoonotic pathogen whose genetic heterogeneity is well known. Five serogroups (A, B, D, E, and F) and 16 serotypes of P. multocida have been recognized thus far based on capsular polysaccharide typing and lipopolysaccharide typing, respectively. Progressive atrophic rhinitis in domestic pigs is caused by P. multocida strains containing toxA, which encodes a 146 kDa heat-labile toxin. Among the five serogroups, only some strains of serogroups A and D are toxigenic. In this study, by comparative analyses of the genomes of many strains, it has been shown that toxA is sparsely distributed in P. multocida. Furthermore, full-length homologs of P. multocida toxA were found only in two other bacterial species. It has also been shown that toxA is usually associated with a prophage, and that some strains contain an orthologous prophage but not toxA. Among the toxA-containing prophages that were compared, an operon putatively encoding a type II restriction-modification system was present only in strains LFB3, HN01, and HN06. These results indicate that the selection and maintenance of the heat-labile toxin and the type II restriction-modification system are evolutionarily less favorable among P. multocida strains. Phylogenetic analysis using the alignment- and parameter-free method CVTree3 showed that deduced proteome sequences can be used as effectively as whole/core genome single nucleotide polymorphisms to group P. multocida strains in relation to their serotypes and (or) genotypes. It remains to be determined if the toxA-containing prophages in strains HN01 and HN06 are inducible, and if they can be used for lysogenic transfer of toxA to other bacteria.

The Role of luxS in Histophilus somni Virulence and Biofilm Formation.

S-Ribosylhomocysteinase (LuxS) is required for the synthesis of the autoinducer-2 (AI-2) quorum-sensing signaling molecule in many Gram-negative bacteria. The bovine (and ovine) opportunistic pathogen Histophilus somni contains luxS and forms a biofilm containing an exopolysaccharide (EPS) in the matrix. Since biofilm formation is regulated by quorum sensing in many bacteria, the roles of luxS in H. somni virulence and biofilm formation were investigated. Although culture supernatants from H. somni were ineffective at inducing bioluminescence in the Vibrio harveyi reporter strain BB170, H. somniluxS complemented the biosynthesis of AI-2 in the luxS-deficient Escherichia coli strain DH5α. H. somni strain 2336 luxS was inactivated by transposon mutagenesis. RNA expression profiles revealed that many genes were significantly differentially expressed in the luxS mutant compared to that in the wild-type, whether the bacteria were grown planktonically or in a biofilm. Furthermore, the luxS mutant had a truncated and asialylated lipooligosaccharide (LOS) and was substantially more serum sensitive than the wild-type. Not surprisingly, the luxS mutant was attenuated in a mouse model for H. somni virulence, and some of the altered phenotypes were partially restored after the mutation was complemented with a functional luxS However, no major differences were observed between the wild-type and the luxS mutant in regard to outer membrane protein profiles, biofilm formation, EPS production, or intracellular survival. These results indicate that luxS plays a role in H. somni virulence in the context of LOS biosynthesis but not biofilm formation or other phenotypic properties examined.

Phylogenetic insights into the diversity of Chryseobacterium species.

The genus Chryseobacterium was formally established in 1994 and contains 112 species with validly published names. Most of these species are yellow or orange coloured, and contain a flexirubin-type pigment. The genomes of 83 of these 112 species have been sequenced in view of their importance in clinical microbiology and potential applications in biotechnology. The National Center for Biotechnology Information taxonomy browser lists 1415 strains as members of the genus Chryseobacterium , of which the genomes of 94 strains have been sequenced. In this study, by comparing the 16S rDNA and the deduced proteome sequences, at least 20 of these strains have been proposed to represent novel species of the genus Chryseobacterium . Furthermore, a yellow-coloured bacterium isolated from dry soil in the USA (and identified as Flavobacterium sp. strain B-14859) has also been reconciled as a novel member of the genus Chryseobacterium based on the analysis of 16S rDNA sequences and the presence of flexirubin. Yet another bacterium (isolated from a water sample collected in the Western Ghats of India and identified as Chryseobacterium sp. strain WG4) was also found to represent a novel species. These proposals need to be validated using polyphasic taxonomic approaches.

Characterization of blaCTX-M sequences of Indian origin and thirteen uropathogenic Escherichia coli isolates resistant to multiple antibiotics.

OBJECTIVES: ESBL-producing isolates of the Enterobacteriaceae occur throughout the world. The objectives of this study were to characterize uropathogenic Escherichia coli isolated at a tertiary care hospital in southern India, and shed light on blaCTX-M sequences of Indian origin. RESULTS: A cohort of 13 urinary isolates of E. coli (obtained from patients at the Sri Sathya Sai Institute of Higher Medical Sciences, Prasanthigram, Andhra Pradesh, India) were characterized and found to be resistant to multiple antibiotics, including extended-spectrum cephalosporins. All 13 isolates contained blaCTX-M-15, and many of them transferred this genotype to at least one laboratory strain of E. coli after conjugation. Analyses of blaCTX-M-15 sequences (n = 141) of Indian origin showed that > 85% of them were obtained from bacteria not associated with the urinary tract, and that E. coli isolates account for majority of all blaCTX-M-15-carrying bacteria reported from India. Other types of blaCTX-M appear to be rare in India, since only six such sequences were reported as of July 2015. The results indicate that 'selection pressure' exerted by extended-spectrum cephalosporins may have stabilized the blaCTX-M-15 genotype among E. coli in India. The rarity of other blaCTX-M suggests that they lack the survival advantage that blaCTX-M-15 may have.

Detection of Antibiotic Resistance Determinants and Their Transmissibility among Clinically Isolated Carbapenem-Resistant Escherichia coli from South India.

OBJECTIVES: The aim of this study was to analyze the prevalence of the CTX-M, TEM, SHV, VIM, NDM, and OXA genes in carbapenemase-producing Escherichia coli and their transmissibility at a tertiary care hospital in south India. MATERIALS AND METHODS: Twenty-one carbapenem-resistant E. coli (carbapenem-resistant Enterobacteriaceae; CRE) were collected from the Sri Sathya Sai Institute of Higher Medical Sciences (Puttaparthi India). Resistance to antibiotics was analyzed by Vitek-2, and the identity of the isolates was confirmed by 16S rDNA sequencing. RAPD and enterobacterial repetitive intergenic consensus (ERIC)-PCR were performed for molecular typing. Metallo-ß-lactamase production was confirmed by a double disc synergy test. The presence of the extended-spectrum ß-lactamases CTX-M, TEM, and SHV and of the carbapenemases NDM, VIM, and OXA was determined by PCR. Carbapenemase variants were further confirmed by sequencing. The transmissibility of the genes was tested by conjugation. RESULTS: Twelve of the 21 (57%) carbapenem-resistant E. coli isolates were community acquired, indicating the spread of CRE in environmental samples. TEM and NDM-5 were found to be the major ß-lactamases produced by the pathogens. OXA-181 was found in 5 of the isolates. All 21 isolates were found to harbor more than one of the tested ß-lactamases, and all of the isolates were found to have the capacity to participate in conjugation; 15 of the transconjugants were found to have acquired the tested ß-lactamases, substantiating their ability to be transferred to other strains of bacteria. CONCLUSION: Monitoring of community-acquired carbapenem-resistant bacteria is very important as the association of resistance determinants with mobile genetic elements would present a serious clinical challenge.

Genomewide characterisation of the genetic diversity of carotenogenesis in bacteria of the order Sphingomonadales.

The order Sphingomonadales is a taxon of bacteria with a variety of physiological features and carotenoid pigments. Some of the coloured strains within this order are known to be aerobic anoxygenic phototrophs that contain characteristic photosynthesis gene clusters (PGCs). Previous work has shown that majority of the ORFs putatively involved in the biosynthesis of C40 carotenoids are located outside the PGCs in these strains. The main purpose of this study was to understand the genetic basis for the various colour/carotenoid phenotypes of the strains of Sphingomonadales. Comparative analyses of the genomes of 41 strains of this order revealed that there were different patterns of clustering of carotenoid biosynthesis (crt) ORFs, with four ORF clusters being the most common. The analyses also revealed that co-occurrence of crtY and crtI is an evolutionarily conserved feature in Sphingomonadales and other carotenogenic bacteria. The comparisons facilitated the categorisation of bacteria of this order into four groups based on the presence of different crt ORFs. Yellow coloured strains most likely accumulate nostoxanthin, and contain six ORFs (group I: crtE, crtB, crtI, crtY, crtZ, crtG). Orange coloured strains may produce adonixanthin, astaxanthin, canthaxanthin and erythroxanthin, and contain seven ORFs (group II: crtE, crtB, crtI, crtY, crtZ, crtG, crtW). Red coloured strains may accumulate astaxanthin, and contain six ORFs (group III: crtE, crtB, crtI, crtY, crtZ, crtW). Non-pigmented strains may contain a smaller subset of crt ORFs, and thus fail to produce any carotenoids (group IV). The functions of many of these ORFs remain to be characterised.

"On demand" redox buffering by H2S contributes to antibiotic resistance revealed by a bacteria-specific H2S donor.

Understanding the mechanisms of antimicrobial resistance (AMR) will help launch a counter-offensive against human pathogens that threaten our ability to effectively treat common infections. Herein, we report bis(4-nitrobenzyl)sulfanes, which are activated by a bacterial enzyme to produce hydrogen sulfide (H2S) gas. We found that H2S helps maintain redox homeostasis and protects bacteria against antibiotic-triggered oxidative stress "on demand", through activation of alternate respiratory oxidases and cellular antioxidants. We discovered, a hitherto unknown role for this gas, that chemical inhibition of H2S biosynthesis reversed antibiotic resistance in multidrug-resistant (MDR) uropathogenic Escherichia coli strains of clinical origin, whereas exposure to the H2S donor restored drug tolerance. Together, our study provides a greater insight into the dynamic defence mechanisms of this gas, modes of antibiotic action as well as resistance while progressing towards new pharmacological targets to address AMR.

Histophilus somni Genomics and Genetics.

Histophilus somni is a commensal and an opportunistic bacterial pathogen associated with multisystemic diseases in cattle and sheep. Some strains of H. somni isolated from the genital tract of cattle are biochemically and serologically similar to the pathogenic strains, but are relatively innocuous. Several virulence factors/mechanisms have been identified in H. somni, of which the phase-variable lipooligosaccharide, induction of apoptosis of host cells, intraphagocytic survival, and immunoglobulin Fc-binding proteins have been well characterized. The genomes of H. somni pneumonia strain 2336 and preputial strain 129Pt have also been sequenced, and comparative analyses of these genomes have provided novel insights into the role of horizontal gene transfer in the evolution of the respective strains. Continued analyses of the genomes of H. somni strains and comparing them to the newly sequenced genomes of other bacteria facilitated the identification of a putative integrative and conjugative element (designated ICEHso2336) encoding tetracycline resistance. Comparative genomics also showed that the uptake signal sequence (5'-AAGTGCGGT) of Haemophilus influenzae is abundant in H. somni and provided a genetic basis for the recalcitrance of some strains of this species to natural transformation. The post-genomic era for H. somni offered an opportunity for the functional characterization of genes identified by computational methods. This opportunity has been realized to a great extent by transcriptomic studies that have identified several small noncoding RNAs and new genes. These new discoveries and developments are expected to stimulate further in-depth investigations of H. somni, especially from the systems biology viewpoint.

Comparative analyses of a putative Francisella conjugative element.

A large circular plasmid detected in Francisella novicida-like strain PA10-7858, designated pFNPA10, was sequenced completely and analyzed. This 41,013-bp plasmid showed no homology to any of the previously sequenced Francisella plasmids and was 8-10 times larger in size than them. A total of 57 ORFs were identified within pFNPA10 and at least 9 of them encoded putative proteins with homology to different conjugal transfer proteins. The presence of iteron-like direct repeats and an ORF encoding a putative replication protein within pFNPA10 suggested that it replicated by the theta mode. Phylogenetic analyses indicated that pFNPA10 had no near neighbors in the databases and that it may have originated within an environmental Francisella lineage. Based on its features, pFNPA10 appears to be a novel extra-chromosomal genetic element within the genus Francisella. The suitability of pFNPA10 as a vector for transformation of species of Francisella by conjugation remains to be explored.

Dehalogenimonas lykanthroporepellens BL-DC-9T simultaneously transcribes many rdhA genes during organohalide respiration with 1,2-DCA, 1,2-DCP, and 1,2,3-TCP as electron acceptors.

The genome sequence of the organohalide-respiring bacterium Dehalogenimonas lykanthroporepellensBL-DC-9(T) contains numerous loci annotated as reductive dehalogenase homologous (rdh) genes based on inferred protein sequence identity with functional dehalogenases of other bacterial species. Many of these genes are truncated, lack adjacent regulatory elements, or lack cognate genes coding for membrane-anchoring proteins typical of the functionally characterized active reductive dehalogenases of organohalide-respiring bacteria. To investigate the expression patterns of the rdh genes in D. lykanthroporepellensBL-DC-9(T), oligonucleotide primers were designed to uniquely target 25 rdh genes present in the genome as well as four putative regulatory genes. RNA extracts from cultures of strain BL-DC-9(T) actively dechlorinating three different electron acceptors, 1,2-dichloroethane, 1,2-dichloropropane, and 1,2,3-trichloropropane were reverse-transcribed and subjected to PCR amplification using rdh-specific primers. Nineteen rdh gene transcripts, including 13 full-length rdhA genes, six truncated rdhA genes, and five rdhA genes having cognate rdhB genes were consistently detected during the dechlorination of all three of the polychlorinated alkanes tested. Transcripts from all four of the putative regulatory genes were also consistently detected. Results reported here expand the diversity of bacteria known to simultaneously transcribe multiple rdh genes and provide insights into the transcription factors associated with rdh gene expression.

Genetic diversity within the genus Francisella as revealed by comparative analyses of the genomes of two North American isolates from environmental sources.

BACKGROUND: Francisella tularensis is an intracellular pathogen that causes tularemia in humans and the public health importance of this bacterium has been well documented in recent history. Francisella philomiragia, a distant relative of F. tularensis, is thought to constitute an environmental lineage along with Francisella novicida. Nevertheless, both F. philomiragia and F. novicida have been associated with human disease, primarily in immune-compromised individuals. To understand the genetic relationships and evolutionary contexts among different lineages within the genus Francisella, the genome of Francisella spp. strain TX07-7308 was sequenced and compared to the genomes of F. philomiragia strains ATCC 25017 and 25015, F. novicida strain U112, and F. tularensis strain Schu S4. RESULTS: The size of strain ATCC 25017 chromosome was 2,045,775 bp and contained 1,983 protein-coding genes. The size of strain TX07-7308 chromosome was 2,035,931 bp and contained 1,980 protein-coding genes. Pairwise BLAST comparisons indicated that strains TX07-7308 and ATCC 25017 contained 1,700 protein coding genes in common. NUCmer analyses revealed that the chromosomes of strains TX07-7308 and ATCC 25017 were mostly collinear except for a few gaps, translocations, and/or inversions. Using the genome sequence data and comparative analyses with other members of the genus Francisella (e.g., F. novicida strain U112 and F. tularensis strain Schu S4), several strain-specific genes were identified. Strains TX07-7308 and ATCC 25017 contained an operon with six open reading frames encoding proteins related to enzymes involved in thiamine biosynthesis that was absent in F. novicida strain U112 and F. tularensis strain Schu S4. Strain ATCC 25017 contained an operon putatively involved in lactose metabolism that was absent in strain TX07-7308, F. novicida strain U112, and F. tularensis strain Schu S4. In contrast, strain TX07-7308 contained an operon putatively involved in glucuronate metabolism that was absent in the genomes of strain ATCC 25017, F. novicida strain U112, and F. tularensis strain Schu S4. The polymorphic nature of polysaccharide biosynthesis/modification gene clusters among different Francisella strains was also evident from genome analyses. CONCLUSIONS: From genome comparisons, it appeared that genes encoding novel functions have contributed to the metabolic enrichment of the environmental lineages within the genus Francisella. The inability to acquire new genes coupled with the loss of ancestral traits and the consequent reductive evolution may be a cause for, as well as an effect of, niche selection of F. tularensis. Sequencing and comparison of the genomes of more isolates are required to obtain further insights into the ecology and evolution of different species within the genus Francisella.

Complete genome sequence of Dehalogenimonas lykanthroporepellens type strain (BL-DC-9(T)) and comparison to "Dehalococcoides" strains.

Dehalogenimonas lykanthroporepellens is the type species of the genus Dehalogenimonas, which belongs to a deeply branching lineage within the phylum Chloroflexi. This strictly anaerobic, mesophilic, non spore-forming, Gram-negative staining bacterium was first isolated from chlorinated solvent contaminated groundwater at a Superfund site located near Baton Rouge, Louisiana, USA. D. lykanthroporepellens was of interest for genome sequencing for two reasons: (a) an unusual ability to couple growth with reductive dechlorination of environmentally important polychlorinated aliphatic alkanes and (b) a phylogenetic position that is distant from previously sequenced bacteria. The 1,686,510 bp circular chromosome of strain BL-DC-9(T) contains 1,720 predicted protein coding genes, 47 tRNA genes, a single large subunit rRNA (23S-5S) locus, and a single, orphan, small subunit rRNA (16S) locus.

A comparative genomics perspective on the genetic content of the alkaliphilic haloarchaeon Natrialba magadii ATCC 43099T.

BACKGROUND: Natrialba magadii is an aerobic chemoorganotrophic member of the Euryarchaeota and is a dual extremophile requiring alkaline conditions and hypersalinity for optimal growth. The genome sequence of Nab. magadii type strain ATCC 43099 was deciphered to obtain a comprehensive insight into the genetic content of this haloarchaeon and to understand the basis of some of the cellular functions necessary for its survival. RESULTS: The genome of Nab. magadii consists of four replicons with a total sequence of 4,443,643 bp and encodes 4,212 putative proteins, some of which contain peptide repeats of various lengths. Comparative genome analyses facilitated the identification of genes encoding putative proteins involved in adaptation to hypersalinity, stress response, glycosylation, and polysaccharide biosynthesis. A proton-driven ATP synthase and a variety of putative cytochromes and other proteins supporting aerobic respiration and electron transfer were encoded by one or more of Nab. magadii replicons. The genome encodes a number of putative proteases/peptidases as well as protein secretion functions. Genes encoding putative transcriptional regulators, basal transcription factors, signal perception/transduction proteins, and chemotaxis/phototaxis proteins were abundant in the genome. Pathways for the biosynthesis of thiamine, riboflavin, heme, cobalamin, coenzyme F420 and other essential co-factors were deduced by in depth sequence analyses. However, approximately 36% of Nab. magadii protein coding genes could not be assigned a function based on Blast analysis and have been annotated as encoding hypothetical or conserved hypothetical proteins. Furthermore, despite extensive comparative genomic analyses, genes necessary for survival in alkaline conditions could not be identified in Nab. magadii. CONCLUSIONS: Based on genomic analyses, Nab. magadii is predicted to be metabolically versatile and it could use different carbon and energy sources to sustain growth. Nab. magadii has the genetic potential to adapt to its milieu by intracellular accumulation of inorganic cations and/or neutral organic compounds. The identification of Nab. magadii genes involved in coenzyme biosynthesis is a necessary step toward further reconstruction of the metabolic pathways in halophilic archaea and other extremophiles. The knowledge gained from the genome sequence of this haloalkaliphilic archaeon is highly valuable in advancing the applications of extremophiles and their enzymes.

Horizontal gene transfer in Histophilus somni and its role in the evolution of pathogenic strain 2336, as determined by comparative genomic analyses.

BACKGROUND: Pneumonia and myocarditis are the most commonly reported diseases due to Histophilus somni, an opportunistic pathogen of the reproductive and respiratory tracts of cattle. Thus far only a few genes involved in metabolic and virulence functions have been identified and characterized in H. somni using traditional methods. Analyses of the genome sequences of several Pasteurellaceae species have provided insights into their biology and evolution. In view of the economic and ecological importance of H. somni, the genome sequence of pneumonia strain 2336 has been determined and compared to that of commensal strain 129Pt and other members of the Pasteurellaceae. RESULTS: The chromosome of strain 2336 (2,263,857 bp) contained 1,980 protein coding genes, whereas the chromosome of strain 129Pt (2,007,700 bp) contained only 1,792 protein coding genes. Although the chromosomes of the two strains differ in size, their average GC content, gene density (total number of genes predicted on the chromosome), and percentage of sequence (number of genes) that encodes proteins were similar. The chromosomes of these strains also contained a number of discrete prophage regions and genomic islands. One of the genomic islands in strain 2336 contained genes putatively involved in copper, zinc, and tetracycline resistance. Using the genome sequence data and comparative analyses with other members of the Pasteurellaceae, several H. somni genes that may encode proteins involved in virulence (e.g., filamentous haemaggutinins, adhesins, and polysaccharide biosynthesis/modification enzymes) were identified. The two strains contained a total of 17 ORFs that encode putative glycosyltransferases and some of these ORFs had characteristic simple sequence repeats within them. Most of the genes/loci common to both the strains were located in different regions of the two chromosomes and occurred in opposite orientations, indicating genome rearrangement since their divergence from a common ancestor. CONCLUSIONS: Since the genome of strain 129Pt was ~256,000 bp smaller than that of strain 2336, these genomes provide yet another paradigm for studying evolutionary gene loss and/or gain in regard to virulence repertoire and pathogenic ability. Analyses of the complete genome sequences revealed that bacteriophage- and transposon-mediated horizontal gene transfer had occurred at several loci in the chromosomes of strains 2336 and 129Pt. It appears that these mobile genetic elements have played a major role in creating genomic diversity and phenotypic variability among the two H. somni strains.

Common ancestry and novel genetic traits of Francisella novicida-like isolates from North America and Australia as revealed by comparative genomic analyses.

Francisella novicida is a close relative of Francisella tularensis, the causative agent of tularemia. The genomes of F. novicida-like clinical isolates 3523 (Australian strain) and Fx1 (Texas strain) were sequenced and compared to F. novicida strain U112 and F. tularensis strain Schu S4. The strain 3523 chromosome is 1,945,310 bp and contains 1,854 protein-coding genes. The strain Fx1 chromosome is 1,913,619 bp and contains 1,819 protein-coding genes. NUCmer analyses revealed that the genomes of strains Fx1 and U112 are mostly colinear, whereas the genome of strain 3523 has gaps, translocations, and/or inversions compared to genomes of strains Fx1 and U112. Using the genome sequence data and comparative analyses with other members of the genus Francisella, several strain-specific genes that encode putative proteins involved in RTX toxin production, polysaccharide biosynthesis/modification, thiamine biosynthesis, glucuronate utilization, and polyamine biosynthesis were identified. The RTX toxin synthesis and secretion operon of strain 3523 contains four open reading frames (ORFs) and was named rtxCABD. Based on the alignment of conserved sequences upstream of operons involved in thiamine biosynthesis from various bacteria, a putative THI box was identified in strain 3523. The glucuronate catabolism loci of strains 3523 and Fx1 contain a cluster of nine ORFs oriented in the same direction that appear to constitute an operon. Strains U112 and Schu S4 appeared to have lost the loci for RTX toxin production, thiamine biosynthesis, and glucuronate utilization as a consequence of host adaptation and reductive evolution. In conclusion, comparative analyses provided insights into the common ancestry and novel genetic traits of these strains.

A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production.

BACKGROUND: Succinate is produced petrochemically from maleic anhydride to satisfy a small specialty chemical market. If succinate could be produced fermentatively at a price competitive with that of maleic anhydride, though, it could replace maleic anhydride as the precursor of many bulk chemicals, transforming a multi-billion dollar petrochemical market into one based on renewable resources. Actinobacillus succinogenes naturally converts sugars and CO2 into high concentrations of succinic acid as part of a mixed-acid fermentation. Efforts are ongoing to maximize carbon flux to succinate to achieve an industrial process. RESULTS: Described here is the 2.3 Mb A. succinogenes genome sequence with emphasis on A. succinogenes's potential for genetic engineering, its metabolic attributes and capabilities, and its lack of pathogenicity. The genome sequence contains 1,690 DNA uptake signal sequence repeats and a nearly complete set of natural competence proteins, suggesting that A. succinogenes is capable of natural transformation. A. succinogenes lacks a complete tricarboxylic acid cycle as well as a glyoxylate pathway, and it appears to be able to transport and degrade about twenty different carbohydrates. The genomes of A. succinogenes and its closest known relative, Mannheimia succiniciproducens, were compared for the presence of known Pasteurellaceae virulence factors. Both species appear to lack the virulence traits of toxin production, sialic acid and choline incorporation into lipopolysaccharide, and utilization of hemoglobin and transferrin as iron sources. Perspectives are also given on the conservation of A. succinogenes genomic features in other sequenced Pasteurellaceae. CONCLUSIONS: Both A. succinogenes and M. succiniciproducens genome sequences lack many of the virulence genes used by their pathogenic Pasteurellaceae relatives. The lack of pathogenicity of these two succinogens is an exciting prospect, because comparisons with pathogenic Pasteurellaceae could lead to a better understanding of Pasteurellaceae virulence. The fact that the A. succinogenes genome encodes uptake and degradation pathways for a variety of carbohydrates reflects the variety of carbohydrate substrates available in the rumen, A. succinogenes's natural habitat. It also suggests that many different carbon sources can be used as feedstock for succinate production by A. succinogenes.

Comparative analyses of two cryptic plasmids from Haemophilus somnus (Histophilus somni).

Haemophilus somnus is an opportunistic bacterial pathogen capable of causing pneumonia, septicemia, and other systemic infections in bovines. An H. somnus isolate from bovine abortion (strain 649) was found to carry a approximately 1.3 kb plasmid (pHS649) that contained partial homology to two previously sequenced Haemophilus/Histophilus plasmids by BLAST analyses. Sequence analysis of pHS649 identified a putative RepA protein with 48% similarity to the RepA protein of Escherichia coli plasmid pKL1. A approximately 5 kb plasmid (pHS129) from H. somnus preputial isolate 129Pt was also sequenced and found to encode two copies of a putative RepB protein. Whereas pHS649 stably replicated in E. coli DH5alpha, pHS129 did not. Genetic relatedness and possible replication mechanisms of these plasmids are described.