David R. Powell

Construction and analysis of chromosomal Clostridium difficile mutants

Clostridium difficile is an emerging nosocomial pathogen of increasing importance and virulence but our ability to study the molecular mechanisms underlying the pathogenesis of C. difficile‐associated disease has been limited because of a lack of tools for its genetic manipulation. We have now developed a reproducible method for the targeted insertional inactivation of chromosomal C. difficile genes. The approach relies on the observation that an Escherichia coli–Clostridium perfringens shuttle vector is unstable in C. difficile and can be used as a form of conditional lethal vector to deliver gene constructs to the chromosome. We have used this methodology to insertionally inactivate two putative response regulator genes, rgaR and rgbR, which encode proteins with similarity to the toxin gene regulator, VirR, from C. perfringens. Transcriptomic analysis demonstrated that the C. difficile RgaR protein positively regulated four genes, including a putative agrBD operon. The RgaR protein was also purified and shown to bind specifically to sites that contained two consensus VirR boxes located just upstream of the putative promoters of these genes. The development of this methodology will significantly enhance our ability to use molecular approaches to develop a greater understanding of the ability of C. difficile to cause disease.

Effects of temperature on gene expression patterns in Leptospira interrogans serovar Lai as assessed by whole-genome microarrays.

ABSTRACT Leptospirosis is an important zoonosis of worldwide distribution. Humans become infected via exposure to pathogenic Leptospira spp. from infected animals or contaminated water or soil. The availability of genome sequences for Leptospira interrogans, serovars Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor known to affect leptospiral protein expression. Leptospira spp. can grow in artificial media at a range of temperatures reflecting conditions found in the environment and the mammalian host. Therefore, transcriptional changes were compared between cultures grown at 20°C, 30°C, 37°C, and 39°C to represent ambient temperatures in the environment, growth under laboratory conditions, and temperatures in healthy and febrile hosts. Data from direct pairwise comparisons of the four temperatures were consolidated to examine transcriptional changes at two generalized biological conditions representing mammalian physiological temperatures (37°C and 39°C) versus environmental temperatures (20°C and 30°C). Additionally, cultures grown at 30°C then shifted overnight to 37°C were compared with those grown long-term at 30°C and 37°C to identify genes potentially expressed in the early stages of infection. Comparison of data sets from physiological versus environmental experiments with upshift experiments provided novel insights into possible transcriptional changes at different stages of infection. Changes included differential expression of chemotaxis and motility genes, signal transduction systems, and genes encoding proteins involved in alteration of the outer membrane. These findings indicate that temperature is an important factor regulating expression of proteins that facilitate invasion and establishment of disease.

Function of the Cytochrome bc1-aa3 Branch of the Respiratory Network in Mycobacteria and Network Adaptation Occurring in Response to Its Disruption

The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three possible terminal oxidase complexes. The structure and function of the electron transport pathway leading from the menaquinol-menaquinone pool to the cytochrome bc1 complex and terminating in the aa3-type cytochrome c oxidase was characterized. M. smegmatis strains with mutations in the bc1 complex and in subunit II of cyctochome c oxidase were found to be profoundly growth impaired, confirming the importance of this respiratory pathway for mycobacterial growth under aerobic conditions. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB, which encodes the bioenergetically less efficient and microaerobically induced cytochrome bd-type menaquinol oxidase that is required for the growth of M. smegmatis under O2-limiting conditions. Further insights into the adaptation of this organism to rerouting of the electron flux through the branch terminating in the bd-type oxidase were revealed by expression profiling of the bc1-deficient mutant strain using a partial-genome microarray of M. smegmatis that is enriched in essential genes. Although the expression profile was indicative of an increase in the reduced state of the respiratory chain, blockage of the bc1-aa3 pathway did not induce the sentinel genes of M. smegmatis that are induced by oxygen starvation and are regulated by the DosR two-component regulator.

Global Gene Expression in Skeletal Muscle from Well-Trained Strength and Endurance Athletes

PURPOSE We used gene microarray analysis to compare the global expression profile of genes involved in adaptation to training in skeletal muscle from chronically strength-trained (ST), endurance-trained (ET), and untrained control subjects (Con). METHODS Resting skeletal muscle samples were obtained from the vastus lateralis of 20 subjects (Con n = 7, ET n = 7, ST n = 6; trained [TR] groups >8 yr specific training). Total RNA was extracted from tissue for two color microarray analysis and quantative (Q)-PCR. Trained subjects were characterized by performance measures of peak oxygen uptake (V x O 2peak) on a cycle ergometer and maximal concentric and eccentric leg strength on an isokinetic dynamometer. RESULTS Two hundred and sixty-three genes were differentially expressed in trained subjects (ET + ST) compared with Con (P < 0.05), whereas 21 genes were different between ST and ET (P < 0.05). These results were validated by reverse transcriptase polymerase chain reaction for six differentially regulated genes (EIFSJ, LDHB, LMO4, MDH1, SLC16A7, and UTRN. Manual cluster analyses revealed significant regulation of genes involved in muscle structure and development in TR subjects compared with Con (P <or= 0.05) and expression correlated with measures of performance (P < 0.05). ET had increased whereas ST had decreased expression of gene clusters related to mitochondrial/oxidative capacity (P <or= 0.05). These mitochondrial gene clusters correlated with V x O 2peak (P < 0.05). V x O 2peak also correlated with expression of gene clusters that regulate fat and carbohydrate oxidation (P < 0.05). CONCLUSION We demonstrate that chronic training subtly coregulates numerous genes from important functional groups that may be part of the long-term adaptive process to adapt to repeated training stimuli.

Convergent Adaptation in the Dominant Global Hospital Clone ST239 of Methicillin-Resistant Staphylococcus aureus

ABSTRACT Infections caused by highly successful clones of hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) are a major public health burden. The globally dominant sequence type 239 (ST239) HA-MRSA clone has persisted in the health care setting for decades, but the basis of its success has not been identified. Taking a collection of 123 ST239 isolates spanning 32 years, we have used population-based functional genomics to investigate the evolution of this highly persistent and successful clone. Phylogenetic reconstruction and population modeling uncovered a previously unrecognized distinct clade of ST239 that was introduced into Australia from Asia and has perpetuated the epidemic in this region. Functional analysis demonstrated attenuated virulence and enhanced resistance to last-line antimicrobials, the result of two different phenomena, adaptive evolution within the original Australian ST239 clade and the introduction of a new clade displaying shifts in both phenotypes. The genetic diversity between the clades allowed us to employ genome-wide association testing and identify mutations in other essential regulatory systems, including walKR, that significantly associate with and may explain these key phenotypes. The phenotypic convergence of two independently evolving ST239 clades highlights the very strong selective pressures acting on HA-MRSA, showing that hospital environments have favored the accumulation of mutations in essential MRSA genes that increase resistance to antimicrobials, attenuate virulence, and promote persistence in the health care environment. Combinations of comparative genomics and careful phenotypic measurements of longitudinal collections of clinical isolates are giving us the knowledge to intelligently address the impact of current and future antibiotic usage policies and practices on hospital pathogens globally. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for innumerable drug-resistant health care-associated infections globally. This study, the first to investigate the evolutionary response of hospital-associated MRSA (HA-MRSA) over many decades, demonstrates how MRSA can persist in a region through the reintroduction of a previously unrecognized distinct clade. This study also demonstrates the crucial adaptive responses of HA-MRSA to the highly selective environment of the health care system, the evolution of MRSA isolates to even higher levels of antibiotic resistance at the cost of attenuated virulence. However, in vivo persistence is maintained, resulting in a clone of HA-MRSA able to resist almost all antimicrobial agents and still cause invasive disease in the heavily compromised hosts found in modern health care settings. Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for innumerable drug-resistant health care-associated infections globally. This study, the first to investigate the evolutionary response of hospital-associated MRSA (HA-MRSA) over many decades, demonstrates how MRSA can persist in a region through the reintroduction of a previously unrecognized distinct clade. This study also demonstrates the crucial adaptive responses of HA-MRSA to the highly selective environment of the health care system, the evolution of MRSA isolates to even higher levels of antibiotic resistance at the cost of attenuated virulence. However, in vivo persistence is maintained, resulting in a clone of HA-MRSA able to resist almost all antimicrobial agents and still cause invasive disease in the heavily compromised hosts found in modern health care settings.

Comparative analysis of long DNA sequences by per element information content using different contexts

BackgroundFeatures of a DNA sequence can be found by compressing the sequence under a suitable model; good compression implies low information content. Good DNA compression models consider repetition, differences between repeats, and base distributions. From a linear DNA sequence, a compression model can produce a linear information sequence. Linear space complexity is important when exploring long DNA sequences of the order of millions of bases. Compressing a sequence in isolation will include information on self-repetition. Whereas compressing a sequence Y in the context of another X can find what new information X gives about Y. This paper presents a methodology for performing comparative analysis to find features exposed by such models.ResultsWe apply such a model to find features across chromosomes of Cyanidioschyzon merolae. We present a tool that provides useful linear transformations to investigate and save new sequences. Various examples illustrate the methodology, finding features for sequences alone and in different contexts. We also show how to highlight all sets of self-repetition features, in this case within Plasmodium falciparum chromosome 2.ConclusionThe methodology finds features that are significant and that biologists confirm. The exploration of long information sequences in linear time and space is fast and the saved results are self documenting.

Genomic Evidence for a Globally Distributed, Bimodal Population in the Ovine Footrot Pathogen Dichelobacter nodosus

ABSTRACT Footrot is a contagious, debilitating disease of sheep, causing major economic losses in most sheep-producing countries. The causative agent is the Gram-negative anaerobe Dichelobacter nodosus. Depending on the virulence of the infective bacterial strain, clinical signs vary from a mild interdigital dermatitis (benign footrot) to severe underrunning of the horn of the hoof (virulent footrot). The aim of this study was to investigate the genetic relationship between D. nodosus strains of different phenotypic virulences and between isolates from different geographic regions. Genome sequencing was performed on 103 D. nodosus isolates from eight different countries. Comparison of these genome sequences revealed that they were highly conserved, with >95% sequence identity. However, single nucleotide polymorphism analysis of the 31,627 nucleotides that were found to differ in one or more of the 103 sequenced isolates divided them into two distinct clades. Remarkably, this division correlated with known virulent and benign phenotypes, as well as with the single amino acid difference between the AprV2 and AprB2 proteases, which are produced by virulent and benign strains, respectively. This division was irrespective of the geographic origin of the isolates. However, within one of these clades, isolates from different geographic regions generally belonged to separate clusters. In summary, we have shown that D. nodosus has a bimodal population structure that is globally conserved and provide evidence that virulent and benign isolates represent two distinct forms of D. nodosus strains. These data have the potential to improve the diagnosis and targeted control of this economically significant disease. IMPORTANCE The Gram-negative anaerobic bacterium Dichelobacter nodosus is the causative agent of ovine footrot, a disease of major importance to the worldwide sheep industry. The known D. nodosus virulence factors are its type IV fimbriae and extracellular serine proteases. D. nodosus strains are designated virulent or benign based on the type of disease caused under optimal climatic conditions. These isolates have similar fimbriae but distinct extracellular proteases. To determine the relationship between virulent and benign isolates and the relationship of isolates from different geographical regions, a genomic study that involved the sequencing and subsequent analysis of 103 D. nodosus isolates was undertaken. The results showed that D. nodosus isolates are highly conserved at the genomic level but that they can be divided into two distinct clades that correlate with their disease phenotypes and with a single amino acid substitution in one of the extracellular proteases. The Gram-negative anaerobic bacterium Dichelobacter nodosus is the causative agent of ovine footrot, a disease of major importance to the worldwide sheep industry. The known D. nodosus virulence factors are its type IV fimbriae and extracellular serine proteases. D. nodosus strains are designated virulent or benign based on the type of disease caused under optimal climatic conditions. These isolates have similar fimbriae but distinct extracellular proteases. To determine the relationship between virulent and benign isolates and the relationship of isolates from different geographical regions, a genomic study that involved the sequencing and subsequent analysis of 103 D. nodosus isolates was undertaken. The results showed that D. nodosus isolates are highly conserved at the genomic level but that they can be divided into two distinct clades that correlate with their disease phenotypes and with a single amino acid substitution in one of the extracellular proteases.

The transcriptomic response of Acinetobacter baumannii to colistin and doripenem alone and in combination in an in vitro pharmacokinetics/pharmacodynamics model

OBJECTIVES Colistin remains a last-line treatment for MDR Acinetobacter baumannii and combined use of colistin and carbapenems has shown synergistic effects against MDR strains. In order to understand the bacterial responses to these antibiotics, we analysed the transcriptome of A. baumannii following exposure to each. METHODS RNA sequencing was employed to determine changes in the transcriptome following treatment with colistin and doripenem, both alone and in combination, using an in vitro pharmacokinetics (PK)/pharmacodynamics model to mimic the PK of both antibiotics in patients. RESULTS After treatment with colistin (continuous infusion at 2 mg/L), >400 differentially regulated genes were identified, including many associated with outer membrane biogenesis, fatty acid metabolism and phospholipid trafficking. No genes were differentially expressed following treatment with doripenem (Cmax 25 mg/L, t1/2 1.5 h) for 15 min, but 45 genes were identified as differentially expressed after 1 h of growth under this condition. Treatment of A. baumannii with both colistin and doripenem together for 1 h resulted in >450 genes being identified as differentially expressed. More than 70% of these gene expression changes were also observed following colistin treatment alone. CONCLUSIONS These data suggest that colistin causes gross damage to the outer membrane, facilitates lipid exchange between the inner and outer membrane and alters the normal asymmetric outer membrane composition. The transcriptional response to colistin was highly similar to that observed for an LPS-deficient strain, indicating that many of the observed changes are responses to outer membrane instability resulting from LPS loss.

Comparison of the RpoH-Dependent Regulon and General Stress Response in Neisseria gonorrhoeae

In the gammaproteobacteria the RpoH regulon is often equated with the stress response, as the regulon contains many of the genes that encode what have been termed heat shock proteins that deal with the presence of damaged proteins. However, the betaproteobacteria primarily utilize the HrcA repressor protein to control genes involved in the stress response. We used genome-wide transcriptional profiling to compare the RpoH regulon and stress response of Neisseria gonorrhoeae, a member of the betaproteobacteria. To identify the members of the RpoH regulon, a plasmid-borne copy of the rpoH gene was overexpressed during exponential-phase growth at 37 degrees C. This resulted in increased expression of 12 genes, many of which encode proteins that are involved in the stress response in other species. The putative promoter regions of many of these up-regulated genes contain a consensus RpoH binding site similar to that of Escherichia coli. Thus, it appears that unlike other members of the betaproteobacteria, N. gonorrhoeae utilizes RpoH, and not an HrcA homolog, to regulate the stress response. In N. gonorrhoeae exposed to 42 degrees C for 10 min, we observed a much broader transcriptional response involving 37 differentially expressed genes. Genes that are apparently not part of the RpoH regulon showed increased transcription during heat shock. A total of 13 genes were also down-regulated. From these results we concluded that although RpoH acts as the major regulator of protein homeostasis, N. gonorrhoeae has additional means of responding to temperature stress.

Natural Variation Identifies ICARUS1, a Universal Gene Required for Cell Proliferation and Growth at High Temperatures in Arabidopsis thaliana

Plants are highly sensitive to environmental changes and even small variations in ambient temperature have severe consequences on their growth and development. Temperature affects multiple aspects of plant development, but the processes and mechanisms underlying thermo-sensitive growth responses are mostly unknown. Here we exploit natural variation in Arabidopsis thaliana to identify and characterize novel components and processes mediating thermo-sensitive growth responses in plants. Phenotypic screening of wild accessions identified several strains displaying pleiotropic growth defects, at cellular and organism levels, specifically at high ambient temperatures. Positional cloning and characterization of the underlying gene revealed that ICARUS1 (ICA1), which encodes a protein of the tRNAHis guanylyl transferase (Thg1) superfamily, is required for plant growth at high temperatures. Transcriptome and gene marker analyses together with DNA content measurements show that ICA1 loss-of-function results in down regulation of cell cycle associated genes at high temperatures, which is linked with a block in G2/M transition and endoreduplication. In addition, plants with mutations in ICA1 show enhanced sensitivity to DNA damage. Characterization of additional strains that carry lesions in ICA1, but display normal growth, shows that alternative splicing is likely to alleviate the deleterious effects of some natural mutations. Furthermore, analyses of worldwide and regional collections of natural accessions indicate that ICA1 loss-of-function has arisen several times independently, and that these occur at high frequency in some local populations. Overall our results suggest that ICA1-mediated-modulation of fundamental processes such as tRNAHis maturation, modify plant growth responses to temperature changes in a quantitative and reversible manner, in natural populations.