Helicobacter pylori are gram-negative bacteria notable for their high level of genetic diversity and plasticity, features that may play a key role in the organism's ability to colonize the human stomach. Homeologous natural transformation, a key contributor to genomic diversification, has been well-described for H. pylori. To examine the mechanisms involved, we performed restriction analysis and sequencing of recombination products to characterize the length, fragmentation, and position of DNA imported via natural transformation. Our analysis revealed DNA imports of small size (1,300 bp, 95% confidence limits 950–1850 bp) with instances of substantial asymmetry in relation to selectable antibiotic-resistance markers. We also observed clustering of imported DNA endpoints, suggesting a possible role for restriction endonucleases in limiting recombination length. Additionally, we observed gaps in integrated DNA and found evidence suggesting that these gaps are the result of two or more separate strand invasions. Taken together, these observations support a system of highly efficient short-fragment recombination involving multiple recombination events within a single locus.
Helicobacter pylori are gram-negative bacteria that have been implicated in human diseases after decades of persistence in the stomach. Known for its high level of genetic diversity, H. pylori is competent to undergo natural transformation, a process in which donor DNA is integrated into the recipient chromosome. To examine the mechanisms involved, we analyzed the DNA imported via natural transformation in an experimental model system. We found variation in the average length of imported DNA fragments, with asymmetry with respect to a selectable marker. We also found evidence that strain-specific restriction endonucleases may limit recombination length. Additionally, we observed gaps in the integrated DNA and provide evidence that these gaps are the result of separate strand invasions. Together, our observations support a highly efficient system of short-fragment recombination involving multiple recombination events within a small region of the chromosome. This helps explain how bacteria are able to employ genetic recombination to efficiently generate and maintain genomic diversification within the population—a feature that helps H. pylori persistently colonize the harsh environment of the human stomach.
Pathogen evolution and subsequent phenotypic heterogeneity during chronic infection are proposed to enhance Staphylococcus aureus survival during human infection. We tested this theory by genetically and phenotypically characterizing strains with mutations constructed in the mismatch repair (MMR) and oxidized guanine (GO) system, termed mutators, which exhibit increased spontaneous-mutation frequencies. Analysis of these mutators revealed not only strain-dependent increases in the spontaneous-mutation frequency but also shifts in mutational type and hot spots consistent with loss of GO or MMR functions. Although the GO and MMR systems are relied upon in some bacterial species to prevent reactive oxygen species-induced DNA damage, no deficit in hydrogen peroxide sensitivity was found when either of these DNA repair pathways was lost in S. aureus. To gain insight into the contribution of increased mutation supply to S. aureus pathoadaptation, we measured the rate of α-hemolysin and staphyloxanthin inactivation during serial passage. Detection of increased rates of α-hemolysin and staphyloxanthin inactivation in GO and MMR mutants suggests that these strains are capable of modifying virulence phenotypes implicated in mediating infection. Accelerated derivation of altered virulence phenotypes, combined with the absence of increased ROS sensitivity, highlights the potential of mutators to drive pathoadaptation in the host and serve as catalysts for persistent infections.
Early detection of oral premalignant lesions (OPL) and oral cancers (OC) is critical for improved survival. We evaluated if the addition of autofluorescence visualization (AFV) to conventional white-light examination (WLE) improved the ability to detect OPLs/OCs. Sixty high-risk patients, with suspicious oral lesions or recently diagnosed untreated OPLs/OCs, underwent sequential surveillance with WLE and AFV. Biopsies were obtained from all suspicious areas identified on both examinations (n = 189) and one normal-looking control area per person (n = 60). Sensitivity, specificity, and predictive values were calculated for WLE, AFV, and WLE + AFV. Estimates were calculated separately for lesions classified by histopathologic grades as low-grade lesions, high-grade lesions (HGL), and OCs. Sequential surveillance with WLE + AFV provided a greater sensitivity than WLE in detecting low-grade lesions (75% versus 44%), HGLs (100% versus 71%), and OCs (100% versus 80%). The specificity in detecting OPLs/OCs decreased from 70% with WLE to 38% with WLE + AFV. Thirteen of the 76 additional biopsies (17%) obtained based on AFV findings were HGLs/OCs. Five patients (8%) were diagnosed with a HGL/OC only because of the addition of AFV to WLE. In seven patients, additional HGL/OC foci or wider OC margins were detected on AFV. Additionally, AFV aided in the detection of metachronous HGL/OC in 6 of 26 patients (23%) with a history of previously treated head and neck cancer. Overall, the addition of AFV to WLE improved the ability to detect HGLs/OCs. In spite of the lower specificity, AFV + WLE can be a highly sensitive first-line surveillance tool for detecting OPLs/OCs in high-risk patients.
Enterococcus faecalis is a member of the intestinal and oral microbiota that may affect the etiology of colorectal and oral cancers. The mechanisms by which E. faecalis may contribute to the initiation and progression of these cancers remain uncertain. Epidermal growth factor receptor (EGFR) signaling is postulated to play a crucial role in oral carcinogenesis. A link between E. faecalis and EGFR signaling in oral cancer has not been elucidated. The present study aimed to evaluate the association between E. faecalis and oral cancer and to determine the underlying mechanisms that link E. faecalis to EGFR signaling. We report the high frequency of E. faecalis infection in oral tumors and the clinical association with EGFR activation. Using human oral cancer cells, we support the clinical findings and demonstrate that E. faecalis can induce EGFR activation and cell proliferation. E. faecalis activates EGFR through production of H2O2, a signaling molecule that activates several signaling pathways. Inhibitors of H2O2 (catalase) and EGFR (gefitinib) significantly blocked E. faecalis-induced EGFR activation and cell proliferation. Therefore, E. faecalis infection of oral tumor tissues suggests a possible association between E. faecalis infection and oral carcinogenesis. Interaction of E. faecalis with host cells and production of H2O2 increase EGFR activation, thereby contributing to cell proliferation.
Hypervirulent variants of Klebsiella pneumoniae have been primarily reported in the Asian Pacific Rim, but they are spreading across the globe. We report the sequence of K. pneumoniae strain hvKP1, which caused liver-splenic abscesses in an otherwise healthy 24-year-old from Buffalo, NY, which will assist in determining why these variants are more pathogenic than “classic” K. pneumoniae strains.
Ventilator-associated pneumonia (VAP) is a leading cause of morbidity and mortality in patients hospitalized in intensive care units. Recent studies suggest that dental plaque biofilms serve as a reservoir for respiratory pathogens. The goal of this study was to determine the genetic relationship between strains of respiratory pathogens first isolated from the oral cavity and later isolated from bronchoalveolar lavage fluid from the same patient undergoing mechanical ventilation with suspected VAP.
Plaque and tracheal secretion samples were obtained on the day of hospital admission and every other day thereafter until discharge from the intensive care unit from 100 patients who underwent mechanical ventilation. Bronchoalveolar lavage was performed for 30 patients with suspected VAP. Pulse-field gel electrophoresis and multilocus sequence typing were used to determine the genetic relatedness of strains obtained from oral, tracheal, and bronchoalveolar lavage samples.
Isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter species, and enteric species recovered from plaque from most patients were indistinguishable from isolates recovered from bronchoalveolar lavage fluid (i.e., had >95% similarity of pulse-field gel electrophoresis patterns). Nearly one-half of the Pseudomonas strains showed identical genetic profiles between patients, which suggested a common environmental source of infection.
Respiratory pathogens isolated from the lung are often genetically indistinguishable from strains of the same species isolated from the oral cavity in patients who receive mechanical ventilation who are admitted to the hospital from the community. Thus, dental plaque serves as an important reservoir for respiratory pathogens in patients who undergo mechanical ventilation.
ClinicalTrials.gov identifier: NCT00123123
Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in S. gordonii CH1 cells treated with native amylase relative to those treated with denatured amylase. An abpA-deficient strain of S. gordonii exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed abpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment.
Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage.
We describe the development and application of a pooled Suppression Subtractive Hybridization (PSSH) method to describe differences between the genomic content of a pool of clinical Staphylococcus aureus isolates and a sequenced reference strain. In comparative bacterial genomics, Suppression Subtractive Hybridization (SSH) is normally utilized to compare genomic features or expression profiles of one strain versus another, which limits its ability to analyze communities of isolates. However, a PSSH approach theoretically enables the user to characterize the entirety of gene content unique to a related group of isolates in a single reaction. These unique fragments may then be linked to individual isolates through standard PCR. This method was applied to examine the genomic diversity found in pools of Staphylococcus aureus isolates associated with complicated bacteremia infections leading to endocarditis and osteomyelitis. Across four pools of 10 isolates each, four hundred and twenty nine fragments not found in or significantly divergent from the S. aureus NCTC 8325 reference genome were detected. These fragments could be linked to individual strains within its pool by PCR. This is the first use of PSSH to examine the S. aureus pangenome. We propose that PSSH is a powerful tool for researchers interested in rapidly comparing the genomic content of multiple unstudied isolates.
Staphylococcus aureus; pangenome; subtractive hybridization
The human intestinal microbiota is composed of 1013 to 1014 microorganisms whose collective genome (“microbiome”) contains at least 100 times as many genes as our own genome. We analyzed ~78 million base pairs of unique DNA sequence and 2062 polymerase chain reaction–amplified 16S ribosomal DNA sequences obtained from the fecal DNAs of two healthy adults. Using metabolic function analyses of identified genes, we compared our human genome with the average content of previously sequenced microbial genomes. Our microbiome has significantly enriched metabolism of glycans, amino acids, and xenobiotics; methanogenesis; and 2-methyl-d-erythritol 4-phosphate pathway–mediated biosynthesis of vitamins and isoprenoids. Thus, humans are superorganisms whose metabolism represents an amalgamation of microbial and human attributes.
The composition of the oral microbiota from 10 individuals with healthy oral tissues was determined using culture-independent techniques. From each individual, 26 specimens, each from different oral sites at a single point in time, were collected and pooled. An eleventh pool was constructed using portions of the subgingival specimens from all 10 individuals. The 16S rRNA gene was amplified using broad-range bacterial primers, and clone libraries from the individual and subgingival pools were constructed. From a total of 11 368 high-quality, non-chimeric, near full-length sequences, 247 species-level phylotypes (using a 99% sequence identity threshold) and 9 bacteria phyla were identified. At least 15 bacterial genera were conserved among all 10 individuals, with significant interindividual differences at the species and strain level. Comparisons of these oral bacterial sequences to near full-length sequences found previously in the large intestines and feces of other healthy individuals suggest that the mouth and intestinal tract harbor distinct sets of bacteria. Co-occurrence analysis demonstrated significant segregation of taxa when community membership was examined at the level of genus, but not at the level of species, suggesting that ecologically-significant, competitive interactions are more apparent at a broader taxonomic level than species. This study is one of the more comprehensive, high-resolution analyses of bacterial diversity within the healthy human mouth to date, and highlights the value of tools from macroecology for enhancing our understanding of bacterial ecology in human health.
oral microbiota; ribosomal RNA sequences; human microbial ecology
Nontypeable Haemophilus influenzae colonizes and infects the airways of adults with chronic obstructive pulmonary disease, the fourth most common cause of death worldwide.Thus, H. influenzae, an exclusively human pathogen, has adapted to survive in the hostile environment of the human airways.To characterize proteins expressed by H. influenzae in the airways, a prototype strain was grown in pooled human sputum to simulate conditions in the human respiratory tract.The proteins from whole bacterial cell lysates were solubilized with a strong buffer and then quantitatively cleaned with an optimized precipitation/on-pellet enzymatic digestion procedure.Proteomic profiling was accomplished by Nano-flow liquid chromatography/mass spectroscopy with low void volume and high separation efficiency with a shallow, long gradient.
A total of 1402 proteins were identified with high confidence, including 170 proteins that were encoded by genes that are annotated as conserved hypothetical proteins.Thirty-one proteins were present in greater abundance in sputum-grown conditions at a ratio of > 1.5 compared to chemically defined media.These included 8 anti-oxidant and 5 stress-related proteins, suggesting that expression of antioxidant activity and stress responses is important for survival in the airways.Four proteins involved in uptake of divalent anions and 9 proteins that function in uptake of various molecules were present in greater abundance in sputum-grown conditions.
Proteomic expression profiling of H. influenzae grown in pooled human sputum revealed increased expression of antioxidant, stress-response proteins and cofactor and nutrient uptake systems compared to media grown cells.These observations suggest that H. influenzae adapts to the oxidative and nutritionally limited conditions of the airways in adults with chronic obstructive pulmonary disease by increasing expression of molecules necessary for survival in these conditions.
In dental plaque α-haemolytic streptococci, including Streptococcus gordonii, are considered beneficial for oral health. These organisms produce hydrogen peroxide (H2O2) at concentrations sufficient to kill many oral bacteria. Streptococci do not produce catalase yet tolerate H2O2. We recently demonstrated that coaggregation with Actinomyces naeslundii stabilizes arginine biosynthesis in S. gordonii. Protein arginine residues are sensitive to oxidation by H2O2. Here, the ability of A. naeslundii to protect S. gordonii against self-produced H2O2 was investigated. Coaggregation with A. naeslundii enabled S. gordonii to grow in the absence of arginine, and promoted survival of S. gordonii following growth with or without added arginine. Arginine-replete S. gordonii monocultures contained 20–30 μM H2O2 throughout exponential growth. Actinomyces naeslundii did not produce H2O2 but synthesized catalase, removed H2O2 from coaggregate cultures and decreased protein oxidation in S. gordonii. On solid medium, S. gordonii inhibited growth of A. naeslundii; exogenous catalase overcame this inhibition. In coaggregate cultures, A. naeslundii cell numbers were >90% lower than in monocultures after 24 h. These results indicate that coaggregation with A. naeslundii protects S. gordonii from oxidative damage. However, high cell densities of S. gordonii inhibit A. naeslundii. Therefore, H2O2 may drive these organisms towards an ecologically balanced community in natural dental plaque.
oral streptococci; hydrogen peroxide; Actinomyces naeslundii; metal-catalyzed oxidation; Streptococcus gordonii; catalase
The recent emergence of multidrug resistance (MDR) in Acinetobacter baumannii has raised concern in health care settings worldwide. In order to understand the repertoire of resistance determinants and their organization and origins, we compared the genome sequences of three MDR and three drug-susceptible A. baumannii isolates. The entire MDR phenotype can be explained by the acquisition of discrete resistance determinants distributed throughout the genome. A comparison of closely related MDR and drug-susceptible isolates suggests that drug efflux may be a less significant contributor to resistance to certain classes of antibiotics than inactivation enzymes are. A resistance island with a variable composition of resistance determinants interspersed with transposons, integrons, and other mobile genetic elements is a significant but not universal contributor to the MDR phenotype. Four hundred seventy-five genes are shared among all six clinical isolates but absent from the related environmental species Acinetobacter baylyi ADP1. These genes are enriched for transcription factors and transporters and suggest physiological features of A. baumannii that are related to adaptation for growth in association with humans.
Acinetobacter baumannii is a bacterial pathogen of increasing medical importance. Little is known about its mechanisms of pathogenesis, and safe reliable agents with predictable activity against A. baumannii are presently nonexistent. The availability of relevant animal infection models will facilitate the study of Acinetobacter biology. In this report we tested the hypothesis that the rat pneumonia and soft-tissue infection models that our laboratory had previously used for studies of extraintestinal pathogenic Escherichia coli were clinically relevant for A. baumannii. Advantages of these models over previously described models were that the animals were not rendered neutropenic and they did not receive porcine mucin with bacterial challenge. Using the A. baumannii model pathogen 307-0294 as the challenge pathogen, the pneumonia model demonstrated all of the features of infection that are critical for a clinically relevant model: namely, bacterial growth/clearance, an ensuing host inflammatory response, acute lung injury, and, following progressive bacterial proliferation, death due to respiratory failure. We were also able to demonstrate growth of 307-0294 in the soft-tissue infection model. Next we tested the hypothesis that the soft-tissue infection model could be used to discriminate between the inherent differences in virulence of various A. baumannii clinical isolates. The ability of A. baumannii to grow and/or be cleared in this model was dependent on the challenge strain. We also hypothesized that complement is an important host factor in protecting against A. baumannii infection in vivo. In support of this hypothesis was the observation that the serum sensitivity of various A. baumannii clinical isolates in vitro roughly paralleled their growth/clearance in the soft-tissue infection model in vivo. Lastly we hypothesized that the soft-tissue infection model would serve as an efficient screening mechanism for identifying gene essentiality for drug discovery. Random mutants of 307-0294 were initially screened for lack of growth in human ascites in vitro. Selected mutants were subsequently used for challenge in the soft-tissue infection model to determine if the disrupted gene was essential for growth in vivo. Using this approach, we have been able to successfully identify a number of genes essential for the growth of 307-0294 in vivo. In summary, these models are clinically relevant and can be used to study the innate virulence of various Acinetobacter clinical isolates and to assess potential virulence factors, vaccine candidates, and drug targets in vivo and can be used for pharmacokinetic and chemotherapeutic investigations.
Interactions involving genetically distinct bacteria, for example, between oral streptococci and actinomyces, are central to dental plaque development. A DNA microarray identified Streptococcus gordonii genes regulated in response to coaggregation with Actinomyces naeslundii. The expression of 23 genes changed >3-fold in coaggregates, including that of 9 genes involved in arginine biosynthesis and transport. The capacity of S. gordonii to synthesize arginine was assessed using a chemically defined growth medium. In monoculture, streptococcal arginine biosynthesis was inefficient and streptococci could not grow aerobically at low arginine concentrations. In dual-species cultures containing coaggregates, however, S. gordonii grew to high cell density at low arginine concentrations. Equivalent cocultures without coaggregates showed no growth until coaggregation was evident (9 h). An argH mutant was unable to grow at low arginine concentrations with or without A. naeslundii, indicating that arginine biosynthesis was essential for coaggregation-induced streptococcal growth. Using quantitative reverse transcriptase PCR, the expression of argC, argG, and pyrAb was strongly (10- to 100-fold) up-regulated in S. gordonii monocultures after 3 h of growth when exogenous arginine was depleted. Cocultures without induced coaggregation showed similar regulation. However, within 1 h after coaggregation with A. naeslundii, the expression of argC, argG, and pyrAb in S. gordonii was partially up-regulated although arginine was plentiful, and mRNA levels did not increase further when arginine was diminished. Thus, A. naeslundii stabilizes S. gordonii expression of arginine biosynthesis genes in coaggregates but not cocultures and enables aerobic growth when exogenous arginine is limited.
The human endogenous intestinal microflora is an essential “organ” in providing nourishment, regulating epithelial development, and instructing innate immunity; yet, surprisingly, basic features remain poorly described. We examined 13,355 prokaryotic ribosomal RNA gene sequences from multiple colonic mucosal sites and feces of healthy subjects to improve our understanding of gut microbial diversity. A majority of the bacterial sequences corresponded to uncultivated species and novel microorganisms. We discovered significant intersubject variability and differences between stool and mucosa community composition. Characterization of this immensely diverse ecosystem is the first step in elucidating its role in health and disease.
The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical ∼272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one ∼270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.
The genetic relatedness of the Bacillus anthracis typing phages Gamma and Cherry was determined by nucleotide sequencing and comparative analysis. The genomes of these two phages were identical except at three variable loci, which showed heterogeneity within individual lysates and among Cherry, Wβ, Fah, and four Gamma bacteriophage sequences.
The genome sequence of the hyperthermophilic bacterium Thermotoga maritima MSB8 presents evidence for lateral gene transfer events between bacterial and archaeal species. To estimate the extent of genomic diversity across the order Thermotogales, a comparative genomic hybridization study was initiated to compare nine Thermotoga strains to the sequenced T. maritima MSB8. Many differences could be associated with substrate utilization patterns, which are most likely a reflection of the environmental niche that these individual species occupy. A detailed analysis of some of the predicted variable regions demonstrates many examples of the deletion/insertion of complete cassettes of genes and of gene rearrangements and insertions of DNA within genes, with the C or N terminus being retained. Although the mechanism for gene transfer in this lineage remains to be elucidated, this analysis suggests possible associations with repetitive elements and highlights the possible benefits of rampant genetic exchange to these species.
Staphylococcus aureus is an opportunistic pathogen and the major causative agent of numerous hospital- and community-acquired infections. Staphylococcus epidermidis has emerged as a causative agent of infections often associated with implanted medical devices. We have sequenced the ∼2.8-Mb genome of S. aureus COL, an early methicillin-resistant isolate, and the ∼2.6-Mb genome of S. epidermidis RP62a, a methicillin-resistant biofilm isolate. Comparative analysis of these and other staphylococcal genomes was used to explore the evolution of virulence and resistance between these two species. The S. aureus and S. epidermidis genomes are syntenic throughout their lengths and share a core set of 1,681 open reading frames. Genome islands in nonsyntenic regions are the primary source of variations in pathogenicity and resistance. Gene transfer between staphylococci and low-GC-content gram-positive bacteria appears to have shaped their virulence and resistance profiles. Integrated plasmids in S. epidermidis carry genes encoding resistance to cadmium and species-specific LPXTG surface proteins. A novel genome island encodes multiple phenol-soluble modulins, a potential S. epidermidis virulence factor. S. epidermidis contains the cap operon, encoding the polyglutamate capsule, a major virulence factor in Bacillus anthracis. Additional phenotypic differences are likely the result of single nucleotide polymorphisms, which are most numerous in cell envelope proteins. Overall differences in pathogenicity can be attributed to genome islands in S. aureus which encode enterotoxins, exotoxins, leukocidins, and leukotoxins not found in S. epidermidis.
To provide data necessary to study catabolite-linked transcriptional networks in Thermotoga maritima, we used full-genome DNA microarray analysis of global transcriptional responses to growth on glucose, lactose, and maltose in a chemostat. A much larger number of genes changed expression in cells grown on lactose than on maltose, each relative to genes expressed in cells grown on glucose. Genes encoding putative oligopeptide transporters were often coregulated with adjacent glycosidase-encoding genes. Genes encoding enzymes catalyzing NADH oxidation were up-regulated on both lactose and maltose. Genes involved in iron and sulfur metabolism were differentially expressed in response to lactose. These data help define the sets of coregulated genes and suggest possible functions for their encoded products.
The genomes of three strains of Listeria monocytogenes that have been associated with food-borne illness in the USA were subjected to whole genome comparative analysis. A total of 51, 97 and 69 strain-specific genes were identified in L.monocytogenes strains F2365 (serotype 4b, cheese isolate), F6854 (serotype 1/2a, frankfurter isolate) and H7858 (serotype 4b, meat isolate), respectively. Eighty-three genes were restricted to serotype 1/2a and 51 to serotype 4b strains. These strain- and serotype-specific genes probably contribute to observed differences in pathogenicity, and the ability of the organisms to survive and grow in their respective environmental niches. The serotype 1/2a-specific genes include an operon that encodes the rhamnose biosynthetic pathway that is associated with teichoic acid biosynthesis, as well as operons for five glycosyl transferases and an adenine-specific DNA methyltransferase. A total of 8603 and 105 050 high quality single nucleotide polymorphisms (SNPs) were found on the draft genome sequences of strain H7858 and strain F6854, respectively, when compared with strain F2365. Whole genome comparative analyses revealed that the L.monocytogenes genomes are essentially syntenic, with the majority of genomic differences consisting of phage insertions, transposable elements and SNPs.
The multisubunit protein, dynactin, is a critical component of the cytoplasmic dynein motor machinery. Dynactin contains two distinct structural domains: a projecting sidearm that interacts with dynein and an actin-like minifilament backbone that is thought to bind cargo. Here, we use biochemical, ultrastructural, and molecular cloning techniques to obtain a comprehensive picture of dynactin composition and structure. Treatment of purified dynactin with recombinant dynamitin yields two assemblies: the actin-related protein, Arp1, minifilament and the p150Glued sidearm. Both contain dynamitin. Treatment of dynactin with the chaotropic salt, potassium iodide, completely depolymerizes the Arp1 minifilament to reveal multiple protein complexes that contain the remaining dynactin subunits. The shoulder/sidearm complex contains p150Glued, dynamitin, and p24 subunits and is ultrastructurally similar to dynactin's flexible projecting sidearm. The dynactin shoulder complex, which contains dynamitin and p24, is an elongated, flexible assembly that may link the shoulder/sidearm complex to the Arp1 minifilament. Pointed-end complex contains p62, p27, and p25 subunits, plus a novel actin-related protein, Arp11. p62, p27, and p25 contain predicted cargo-binding motifs, while the Arp11 sequence suggests a pointed-end capping activity. These isolated dynactin subdomains will be useful tools for further analysis of dynactin assembly and function.
The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170’s putative cargo-binding domain. Overexpression studies using p150Glued, the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.