Bordetella pertussis is the causative agent of whooping cough, a highly contagious, acute respiratory illness that has seen resurgence despite the use of vaccines. We present the complete genome sequence of a clinical strain of B. pertussis, D420, which is representative of a currently circulating clade of this pathogen. Copyright © 2015 Boinett et al.
Aliivibrio wodanis and Moritella viscosa have often been isolated concurrently from fish with winter-ulcer disease. Little is known about the interaction between the two bacterial species and how the presence of one bacterial species affects the behaviour of the other.The impact on bacterial growth in co-culture was investigated in vitro, and the presence of A. wodanis has an inhibitorial effect on M. viscosa. Further, we have sequenced the complete genomes of these two marine Gram-negative species, and have performed transcriptome analysis of the bacterial gene expression levels from in vivo samples. Using bacterial implants in the fish abdomen, we demonstrate that the presence of A. wodanis is altering the gene expression levels of M. viscosa compared to when the bacteria are implanted separately.From expression profiling of the transcriptomes, it is evident that the presence of A. wodanis is altering the global gene expression of M. viscosa. Co-cultivation studies showed that A. wodanis is impeding the growth of M. viscosa, and that the inhibitorial effect is not contact-dependent.
Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most serious honey bee brood bacterial disease. We isolated and characterized P. larvae-directed bacteriophages and developed criteria for safe phage therapy. Whole-genome analysis of a highly lytic virus of the family Siphoviridae (HB10c2) provided a detailed safety profile and uncovered its lysogenic nature and a putative beta-lactamase-like protein. To rate its antagonistic activity against the pathogens targeted and to specify potentially harmful effects on the bee population and the environment, P. larvae genotypes ERIC I to IV, representatives of the bee gut microbiota, and a broad panel of members of the order Bacillales were analyzed for phage HB10c2-induced lysis. Breeding assays with infected bee larvae revealed that the in vitro phage activity observed was not predictive of the real-life scenario and therapeutic efficacy. On the basis of the disclosed P. larvae-bacteriophage coevolution, we discuss the future prospects of AFB phage therapy. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
The genome of the soil-dwelling heterotrophic N2-fixing Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 (ATCC 4412) (Ac-8003) has been determined. It consists of 7 circular replicons totalling 5,192,291 bp comprising a circular chromosome of 4,591,803 bp and six plasmids pAcX50a, b, c, d, e, f of 10,435 bp, 13,852, 62,783, 69,713, 132,724, and 311,724 bp respectively. The chromosome has a G+C content of 66.27% and the six plasmids have G+C contents of 58.1, 55.3, 56.7, 59.2, 61.9, and 62.6% respectively. The methylome has also been determined and 5 methylation motifs have been identified. The genome also contains a very high number of transposase/inactivated transposase genes from at least 12 of the 17 recognised insertion sequence families. The Ac-8003 genome has been compared with that of Azotobacter vinelandii ATCC BAA-1303 (Av-DJ), a derivative of strain O, the only other member of the Azotobacteraceae determined so far which has a single chromosome of 5,365,318 bp and no plasmids. The chromosomes show significant stretches of synteny throughout but also reveal a history of many deletion/insertion events. The Ac-8003 genome encodes 4628 predicted protein-encoding genes of which 568 (12.2%) are plasmid borne. 3048 (65%) of these show > 85% identity to the 5050 protein-encoding genes identified in Av-DJ, and of these 99 are plasmid-borne. The core biosynthetic and metabolic pathways and macromolecular architectures and machineries of these organisms appear largely conserved including genes for CO-dehydrogenase, formate dehydrogenase and a soluble NiFe-hydrogenase. The genetic bases for many of the detailed phenotypic differences reported for these organisms have also been identified. Also many other potential phenotypic differences have been uncovered. Properties endowed by the plasmids are described including the presence of an entire aerobic corrin synthesis pathway in pAcX50f and the presence of genes for retro-conjugation in pAcX50c. All these findings are related to the potentially different environmental niches from which these organisms were isolated and to emerging theories about how microbes contribute to their communities.
In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F’-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10-12 CFU/recipient per hour.
Respiratory infectious diseases are the third cause of worldwide death. The nasopharynx is the portal of entry and the ecological niche of many microorganisms, of which some are pathogenic to humans, such as Neisseria meningitidis and Moraxella catarrhalis. These microbes possess several surface structures that interact with the actors of the innate immune system. In our attempt to understand the past evolution of these bacteria and their adaption to the nasopharynx, we first studied differences in cell wall structure, one of the strongest immune-modulators. We were able to show that a modification of peptidoglycan (PG) composition (increased proportion of pentapeptides) and a cell shape change from rod to cocci had been selected for along the past evolution of N. meningitidis. Using genomic comparison across species, we correlated the emergence of the new cell shape (cocci) with the deletion, from the genome of N. meningitidis ancestor, of only one gene: yacF. Moreover, the reconstruction of this genetic deletion in a bacterium harboring the ancestral version of the locus together with the analysis of the PG structure, suggest that this gene is coordinating the transition from cell elongation to cell division. Accompanying the loss of yacF, the elongation machinery was also lost by several of the descendants leading to the change in the PG structure observed in N. meningitidis. Finally, the same evolution was observed for the ancestor of M. catarrhalis. This suggests a strong selection of these genetic events during the colonization of the nasopharynx. This selection may have been forced by the requirement of evolving permissive interaction with the immune system, the need to reduce the cellular surface exposed to immune attacks without reducing the intracellular storage capacity, or the necessity to better compete for adhesion to target cells.
Legionella is the causative agent for Legionnaires’ disease (LD) and is responsible for several large outbreaks in the world. More than 90% of LD cases are caused by Legionella pneumophila, and studies on the origin and transmission routes of this pathogen rely on adequate molecular characterization of isolates. Current typing of L. pneumophila mainly depends on sequence-based typing (SBT). However, studies have shown that in some outbreak situations, SBT does not have sufficient discriminatory power to distinguish between related and nonrelated L. pneumophila isolates. In this study, we used a novel high-resolution typing technique, called whole-genome mapping (WGM), to differentiate between epidemiologically related and nonrelated L. pneumophila isolates. Assessment of the method by various validation experiments showed highly reproducible results, and WGM was able to confirm two well-documented Dutch L. pneumophila outbreaks. Comparison of whole-genome maps of the two outbreaks together with WGMs of epidemiologically nonrelated L. pneumophila isolates showed major differences between the maps, and WGM yielded a higher discriminatory power than SBT. In conclusion, WGM can be a valuable alternative to perform outbreak investigations of L. pneumophila in real time since the turnaround time from culture to comparison of the L. pneumophila maps is less than 24 h. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Multidrug resistance, strong side effects, and compliance problems in TB chemotherapy mandate new ways to kill Mycobacterium tuberculosis (Mtb). Here we show that deletion of the gene encoding homoserine transacetylase (metA) inactivates methionine and S-adenosylmethionine (SAM) biosynthesis in Mtb and renders this pathogen exquisitely sensitive to killing in immunocompetent or immunocompromised mice, leading to rapid clearance from host tissues. Mtb ?metA is unable to proliferate in primary human macrophages, and in vitro starvation leads to extraordinarily rapid killing with no appearance of suppressor mutants. Cell death of Mtb ?metA is faster than that of other auxotrophic mutants (i.e., tryptophan, pantothenate, leucine, biotin), suggesting a particularly potent mechanism of killing. Time-course metabolomics showed complete depletion of intracellular methionine and SAM. SAM depletion was consistent with a significant decrease in methylation at the DNA level (measured by single-molecule real-time sequencing) and with the induction of several essential methyltransferases involved in biotin and menaquinone biosynthesis, both of which are vital biological processes and validated targets of antimycobacterial drugs. Mtb ?metA could be partially rescued by biotin supplementation, confirming a multitarget cell death mechanism. The work presented here uncovers a previously unidentified vulnerability of Mtb-the incapacity to scavenge intermediates of SAM and methionine biosynthesis from the host. This vulnerability unveils an entirely new drug target space with the promise of rapid killing of the tubercle bacillus by a new mechanism of action.
Rhipicephalus microplus, the cattle fever tick, is a global economic problem to the cattle industry due to direct infestation of cattle and pathogens transmitted during feeding. Cattle fever tick outbreaks continue to occur along the Mexico-US border even though the tick has been eradicated from the USA. The organophosphate (OP) coumaphos targets acetylcholinesterase (AChE) and is the approved acaricide for eradicating cattle fever tick outbreaks. There is evidence for coumaphos resistance developing in cattle ticks in Mexico, and OP-resistant R. microplus ticks were discovered in outbreak populations of Texas in 2005. The molecular basis of coumaphos resistance is not known, and our study was established to gather further information on whether AChE1 is involved in the resistance mechanism. We also sought information on allele diversity in tick populations with different levels of coumaphos resistance. The overarching project goal was to define OP resistance-associated gene mutations such that a DNA-based diagnostic assay could be developed to assist the management of resistance. Three different AChE transcripts have been reported in R. microplus, and supporting genomic and transcriptomic data are available at CattleTickBase. Here, we report the complete R. microplus AChE1 gene ascertained by sequencing a bacterial artificial chromosome clone containing the entire coding region and the flanking 5′ and 3′ regions. We also report AChE1 sequences of larval ticks from R. microplus strains having different sensitivities to OP. To accomplish this, we sequenced a 669-bp region of the AChE1 gene corresponding to a 223 amino acid region of exon 2 to assess alleles in seven strains of R. microplus with varying OP resistance phenotypes. We identified 72 AChE1 sequence variants, 2 of which are strongly associated with OP-resistant phenotypes. Esterase-like sequences from the R. microplus transcriptome RmiTr Version 1.0 were compared to the available sequence databases to identify other transcripts with similarity to AChE1.
Haloterrigena jeotgali is a halophilic archaeon within the family Natrialbaceae that was isolated from shrimp jeotgal, a traditional Korean salt-fermented food. A29(T) is the type strain of H. jeotgali, and is a Gram-negative staining, non-motile, rod-shaped archaeon that grows in 10 %-30 % (w/v) NaCl. We present the annotated H. jeotgali A29(T) genome sequence along with a summary of its features. The 4,131,621 bp genome with a GC content of 64.9 % comprises 4,215 protein-coding genes and 127 RNA genes. The sequence can provide useful information on genetic mechanisms that enable haloarchaea to endure a hypersaline environment.
Serotype IV group B Streptococcus (GBS) is emerging in Canada and the United States with rates as high as 5% of the total burden of adult invasive GBS disease. To understand this emergence, we studied the population structure and assessed the antimicrobial susceptibility of serotype IV isolates causing adult invasive infection in Manitoba and Saskatchewan, Canada, between 2010 and 2014. Whole-genome sequencing was used to determine multilocus sequence typing information and identify genes encoding antimicrobial resistance in 85 invasive serotype IV GBS strains. Antimicrobial susceptibility testing was performed by standard methods. Strain divergence was assessed using genome-wide single-nucleotide polymorphism analysis. Serotype IV strains were responsible for 16.9% of adult invasive GBS infections in Manitoba and Saskatchewan during the period. The majority of serotype IV isolates (89%) were clonally related, tetracycline-, erythromycin-, and clindamycin-resistant sequence type 459 (ST459) strains that possessed genes tetM and ermTR. Genome comparisons between ST459 and serotype V ST1 GBS identified several areas of recombination in an overall similar genomic background. Serotype IV ST459 GBS strains are expanding and causing a substantial percentage of adult invasive GBS disease. This emergence may be linked to the acquisition of resistance to tetracycline, macrolides, and lincosamides. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
This month’s Genome Watch looks at how whole-genome sequencing (WGS) can be used to track the source of Pseudomonas aeruginosa infection and to investigate the transition and adaptation of this opportunistic pathogen from the environment to the human host.
As part of a genome sequencing project for Ophiocordyceps sinensis, strain 1229, a complete mitochondrial (mt) genome was assembled as a single circular dsDNA of 157,510?bp, one of the largest reported for fungi. Conserved genes including the large and small rRNA subunits, 27 tRNA and 15 protein-coding genes, were identified. In addition, 58 non-conserved open reading frames (ncORFs) in the intergenic and intronic regions were also identified. Transcription analyses using RNA-Seq validated the expression of most conserved genes and ncORFs. Fifty-two introns (groups I and II) were found within conserved genes, accounting for 68.5% of the genome. Thirty-two homing endonucleases (HEs) with motif patterns LAGLIDADG (21) and GIY-YIG (11) were identified in group I introns. The ncORFs found in group II introns mostly encoded reverse transcriptases (RTs). As in other hypocrealean fungi, gene contents and order were found to be conserved in the mt genome of O. sinensis, but the genome size was enlarged by longer intergenic regions and numerous introns. Intergenic and intronic regions were composed of abundant repetitive sequences usually associated with mobile elements. It is likely that intronic ncORFs, which encode RTs and HEs, may have contributed to the enlarged mt genome of O. sinensis.
Staphylococcus schleiferi, a Gram-positive and coagulase-variable organism, is an opportunistic human pathogen and a major cause of skin and soft tissue infections in dogs. Here, we report the first S. schleiferi genome sequence and methylome from four canine clinical isolates. Copyright © 2015 Misic et al.
Whole-genome sequences for Stenotrophomonas maltophilia serial isolates from a bacteremic patient before and after development of levofloxacin resistance were assembled de novo and differed by one single-nucleotide variant in smeT, a repressor for multidrug efflux operon smeDEF. Along with sequenced isolates from five contemporaneous cases, they displayed considerable diversity compared against all published complete genomes. Whole-genome sequencing and complete assembly can conclusively identify resistance mechanisms emerging in S. maltophilia strains during clinical therapy. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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