Bacterial Life Cycle Research Articles

Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1

Thymidylate synthase X (ThyX) catalyzes the methylation of dUMP to form dTMP in bacterial life cycle and is regarded as a promising target for antibiotics discovery. Helicobacter pylori is a human pathogen associated with a number of human diseases. Here, we cloned and purified the ThyX enzyme from H. pylori SS1 strain (HpThyX). The recombinant HpThyX was discovered to exhibit the maximum activity at pH 8.5, and K(m) values of the two substrates dUMP and CH(2) H(4) folate were determined to be 15.3 ± 1.25 μM and 0.35 ± 0.18 mM, respectively. The analyzed crystal structure of HpThyX with the cofactor FAD and the substrate dUMP (at 2.31 Å) revealed that the enzyme was a tetramer bound to four dUMP and four FAD molecules. Different from the catalytic feature of the classical thymidylate synthase (ThyA), N5 atom of the FAD functioned as a nucleophile in the catalytic reaction instead of Ser84 and Ser85 residues. Our current work is expected to help better understand the structural and enzymatic features of HpThyX thus further providing valuable information for anti-H. pylori inhibitor discovery.

Reference genes for quantitative, reverse-transcription PCR in Bacillus cereus group strains throughout the bacterial life cycle

Quantitative reverse-transcription PCR (RT-qPCR) has become a major tool to better understand the biology and pathogenesis of bacteria. One prerequisite of valid RT-qPCR data is their proper normalization to stably expressed reference genes. To identify and evaluate reference genes suitable for normalization of gene expression data in Bacillus cereus group strains, mRNA levels of eleven candidate reference genes (rpsU, nifU, udp (UDP-N-acetylglucosamine 2-epimerase), BT9727_5154/BC_5475, BT9727_4034/BC_4293, BT9727_4549/BC_4813, pspA, gatB_Yqey (gatB_Yqey domain containing protein), helicase (SWF/SNF family protein), adk and pta) and a target gene (BT9727_3305/BC3547+BC3546) were quantified by RT-qPCR at different time points throughout the entire life cycle of the wild-type B. cereus ATCC 14579 and Bacillus thuringiensis subsp. konkukian 97-27, a phylogenetically closely related strain to Bacillus anthracis. The programs geNorm and Normfinder were used to calculate expression stabilities and identified the genes gatB_Yqey, rpsU and udp as the most stably expressed reference genes. Compared to this combination or the sets of reference genes as recommended by geNorm or Normfinder, normalization using a traditional housekeeping gene (adk) alone resulted in significantly different gene expression results and in a significant overestimation of the target gene transcription. Normalization of the data to the reference gene gatB_Yqey alone showed no or only small differences to the reference gene combinations indicating that gatB_Yqey may be used as a single reference gene when investigating rather large changes in mRNA transcription. Otherwise, a combination of the stably expressed reference genes is recommended. In conclusion, the present study underlines the importance of normalization to stably expressed reference genes and presents valid endogenous controls suitable for normalization of transcriptional data throughout the life cycle of B. cereus group strains.

Correlating in Vitro Measurements of Protein-DNA Binding Affinities with in Vivo Repression and Impact on the Growth Rate of the Host Organism

In order to fully decode the information of patient genomes, we must determine which protein polymorphisms alter function. One resource for addressing this question is the sequence set of naturally occurring homologs. Sequence comparisons easily identify conserved positions, which usually cannot be changed without consequence. However, the impact of changing nonconserved positions is less-easily predicted. We have devised a model system for testing computational identifications of important nonconserved positions and measuring the functional impact from amino acid substitutions at these positions. The model system is based on chimeric proteins of the LacI/GalR family; functional change can be detected as either altered in vivo repression or altered in vitro DNA binding properties. For this system to accurately model a naturally-evolving system, an observed change in repression must be large enough to impact the growth of the host organism. Here, we use a series of point mutations in an engineered LacI/GalR transcription repressor to correlate altered in vivo repression with thermodynamic measurements of DNA binding affinities and bacterial growth rates. Results will determine whether most changes in repression are due to changes in DNA-binding affinity for the in vivo operator DNA binding site, and will determine how large a change in repression is required to alter bacterial life cycles.

The Spatial Architecture of Bacillus subtilis Biofilms Deciphered Using a Surface-Associated Model and In Situ Imaging

The formation of multicellular communities known as biofilms is the part of bacterial life cycle in which bacteria display cooperative behaviour and differentiated phenotypes leading to specific functions. Bacillus subtilis is a Gram-positive bacterium that has served for a decade as a model to study the molecular pathways that control biofilm formation. Most of the data on B. subtilis biofilms have come from studies on the formation of pellicles at the air-liquid interface, or on the complex macrocolonies that develop on semi-solid nutritive agar. Here, using confocal laser scanning microcopy, we show that B. subtilis strains of different origins are capable of forming biofilms on immersed surfaces with dramatically protruding “beanstalk-like” structures with certain strains. Indeed, these structures can reach a height of more than 300 µm with one undomesticated strain from a medical environment. Using 14 GFP-labeled mutants previously described as affecting pellicle or complex colony formation, we have identified four genes whose inactivation significantly impeded immersed biofilm development, and one mutation triggering hyperbiofilm formation. We also identified mutations causing the three-dimensional architecture of the biofilm to be altered. Taken together, our results reveal that B. subtilis is able to form specific biofilm features on immersed surfaces, and that the development of these multicellular surface-associated communities involves regulation pathways that are common to those governing the formation of pellicle and/or complex colonies, and also some specific mechanisms. Finally, we propose the submerged surface-associated biofilm as another relevant model for the study of B. subtilis multicellular communities.

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

BackgroundThe first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.Methods and FindingsWe found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.ConclusionsWe found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.

Peptide deformylase – a promising therapeutic target for tuberculosis and antibacterial drug discovery

Background: Tuberculosis (TB) remains the most important infectious disease causing morbidity and death, due to the human pathogen Mycobacterium tuberculosis. The emergence of multi-drug-resistant and extensively-drug-resistant forms of TB have resulted in an increase in the number of TB cases. Thus, there is an urgent need to identify new drugs with novel targets to ensure future therapeutic success. Studies have indicated that peptide deformylase is an interesting potential candidate for discovering antimicrobial agents. Objective: To explore the role of peptide deformylase, a highly conserved metalloprotease and an essential enzyme in bacterial life cycle, as a target for antibacterial as well as antimycobacterial drug development. Methods: This review is based on recent published literature and online resources related to peptide deformylase inhibitors and their anbacterial potential. Results/conclusion: Peptide deformylase is an emerging therapeutic target for the treatment of tuberculosis and peptide deformylase inhibitors can act as potential future antibacterial agents.

A Role for Single-Stranded Exonucleases in the Use of DNA as a Nutrient

Nutritional competence is the ability of bacterial cells to utilize exogenous double-stranded DNA molecules as a nutrient source. We previously identified several genes in Escherichia coli that are important for this process and proposed a model, based on models of natural competence and transformation in bacteria, where it is assumed that single-stranded DNA (ssDNA) is degraded following entry into the cytoplasm. Since E. coli has several exonucleases, we determined whether they play a role in the long-term survival and the catabolism of DNA as a nutrient. We show here that mutants lacking either ExoI, ExoVII, ExoX, or RecJ are viable during all phases of the bacterial life cycle yet cannot compete with wild-type cells during long-term stationary-phase incubation. We also show that nuclease mutants, alone or in combination, are defective in DNA catabolism, with the exception of the ExoX(-) single mutant. The ExoX(-) mutant consumes double-stranded DNA better than wild-type cells, possibly implying the presence of two pathways in E. coli for the processing of ssDNA as it enters the cytoplasm.

Development of a novel fluorescent substrate for Autolysin E, a bacterial type II amidase

The bifunctional Autolysin E from Staphylococcus epidermidis, contains a Zn 2+-dependent N-acetylmuramoyl- l-alanine amidase AmiE (EC 3.5.1.28). This enzyme hydrolyzes the amide bond between the carbohydrate chain and the peptide stem of bacterial peptidoglycan. Since peptidoglycan is the mayor component of bacterial cell walls, type II amidases like Autolysin E play an essential role in the bacterial life cycle. Therefore bacterial amidases are appropriate drug targets in the development of antibiotics. The drug discovery process relies on sensitive enzyme assay systems to test possible lead candidates for enzyme inhibition. However, specific determination of bacterial amidase activity is complicated because a simple and accurate enzyme assay is currently unavailable. In this study we developed a sensitive fluorescent substrate for the type II amidase Autolysin E from S. epidermidis, which is suitable for quantifying amidase activity in a high throughput compatible fashion. Using derivatives of the substrate Mca-Ala- d-isoGln-Lys(Dnp)- d-Ala-Arg-OH, we were further able to characterize the amidase substrate specificity of Autolysin E.

3-Mercaptopropionate Dioxygenase, a Cysteine Dioxygenase Homologue, Catalyzes the Initial Step of 3-Mercaptopropionate Catabolism in the 3,3-Thiodipropionic Acid-degrading Bacterium Variovorax paradoxus

The thioether 3,3-thiodipropionic acid can be used as precursor substrate for biotechnological synthesis of 3-mercaptopropionic acid-containing polythioesters. Therefore, the hitherto unknown catabolism of this compound was elucidated to engineer novel and improved polythioester biosynthesis pathways in the future. Bacteria capable of using 3,3-thiodipropionic acid as the sole source of carbon and energy for growth were enriched from the environment. From eleven isolates, TBEA3, TBEA6, and SFWT were morphologically and physiologically characterized. Their 16 S rDNAs and other features affiliated these isolates to the beta-subgroup of the proteobacteria. Tn5::mob mutagenesis of isolate Variovorax paradoxus TBEA6 yielded ten mutants fully or partially impaired in growth on 3,3-thiodipropionic acid. Genotypic characterization of two 3,3-thiodipropionic acid-negative mutants demonstrated the involvement of a bacterial cysteine dioxygenase (EC 1.13.11.22) homologue in the further catabolism of the 3,3-thiodipropionic acid cleavage product 3-mercaptopropionic acid. Detection of 3-sulfinopropionate in the supernatant of one of these mutants during cultivation on 3,3-thiodipropionic acid as well as in vivo and in vitro enzyme assays using purified protein demonstrated oxygenation of 3-mercaptopropionic acid to 3-sulfinopropionate by this enzyme; cysteine and cysteamine were not used as substrate. Beside cysteine dioxygenase and cysteamine dioxygenase, this 3-mercaptopropionic acid dioxygenase is the third example for a thiol dioxygenase and the first report about the microbial catabolism of 3-mercaptopropionic acid. Insertion of Tn5::mob in a gene putatively coding for a family III acyl-CoA-transferase resulted in the accumulation of 3-sulfinopropionate during cultivation on 3,3-thiodipropionic acid, indicating that this compound is further metabolized to 3-sulfinopropionyl-CoA and subsequently to propionyl-CoA.

Not so simple after all

Biologists have long thought that the internal workings of prokaryotes—the smallest and simplest organisms, including bacteria—are well understood, and have accordingly considered eukaryotic organisms and their cells to be more fascinating objects for study. During the past decade, however, the focus of some research has begun to turn back to prokaryotes—particularly because genomic analyses have shown the enormous flexibility and adaptability of these seemingly simple organisms. Moreover, recent structural research has shown that the cell components of prokaryotes might also be more complex than previously thought. It has long been held that prokaryotic cells lack the internal structure and organization of eukaryotic cells, which have various membrane‐bound organelles, such as mitochondria, chloroplasts and the nucleus. Indeed, prokaryotes are assumed to be the predecessors of some eukaryotic organelles—permanently captured after a period of endosymbiosis. > …there is no rule that says an organelle must be enclosed by a membrane The view that bacterial cells are structurally simpler than those of ‘higher’ organisms is reflected in many text books and web sites with comments such as: “the ribosome is the only prokaryotic organelle”—if, indeed, this highly complex and sophisticated protein‐synthesizing machinery is an organelle in its own right. These types of statement are correct in the sense that prokaryotes contain no membrane‐bound organelles; but there is no rule that says an organelle must be enclosed by a membrane. Many eukaryotic ribosomes are freely suspended in the cytoplasm where they manufacture cytosolic proteins. Furthermore, the word ‘organelle’ itself is being replaced by the terms ‘functional unit’ or ‘compartment’, which are increasingly used to describe coherent structures within a cell that perform clearly defined sets of tasks—such as mitochondria, which create energy by aerobic respiration, or chloroplasts, which do so by photosynthesis. By using this definition, a series of recent discoveries show that prokaryotes also …

Stimulation of biofilm formation by insertion of Tetrahymena pyriformis wells within Burkholderia cenocepacia biofilms

Biofilm formation is an important part of the bacterial life cycle. Biofilms provide bacterial resistance to external stresses and protozoan grazing. Biofilm formation by the wild type of B. cenocepacia strain 370 in the presence of the free-living ciliate Tetrahymena pyriformis was studied. T. pyriformis grazed on planktonic bacteria and reduced the planktonic bacterial subpopulation while it noticeably stimulated biofilm formation. When cultivated alone, T. pyriformis did not form visible biofilms. Confocal laser scanning microscopy was used to demonstrate the inclusion and further destruction of protozoan cells within the biofilms formed by the bacteria. The destruction of protozoan cells was accompanied by the exit of bacteria from vacuoles and intracytoplasmic multiplication; changes in the form of protozoan cells; the demolition of internal structures; and the visual exit of the cytoplasmic content from destructing cells. Microcolonies of a characteristic round shape were revealed in the biofilms formed by B. cenocepacia in the presence of T. pyriformis. These structures were absent in the biofilms formed by B. cenocepacia alone. Insertion of protozoan cells within biofilms seems to be a driving force that promotes biofilm proliferation and influences their structure. The mortality of protozoan cells in the biofilms caused a decrease in the T. pyriformis population under conditions advantageous to B. cenocepacia biofilm formation. The mutant B. cenocepacia strain Bcb-1, which is unable to form biofilms, was isolated by plasposon mutagenesis. In contrast to the parental strain, the cocultivation with Bcb-1 bacteria improved the growth of T. pyriformis. A mutation was mapped in the ompR gene.

In vivocolonization profile study ofBordetella bronchisepticain the nasal cavity

Bordetella bronchiseptica chronically infects a wide range of mammals, and resides primarily in the nasal cavity of the infected host. Multiple virulence factors of Bordetella species have been studied in the context of lower respiratory tract infections, but relatively less is known about the bacterial life cycle in the nasal cavity. Evidences were discovered for Bvg intermediate (Bvgi) phase expression in vivo and that the major adhesin filamentous hemagglutinin plays a major role in the colonization of B. bronchiseptica in the unciliated olfactory epithelia of the nasal cavity.

Novel Genetic Techniques and Approaches in the Microbial Genomics Era

The availability of microbial genome sequences has ushered in the genomics era and has led to numerous technical advancements over the past decade. These advances have been both in the bioinformatics field that has integrated computer-based approaches with biology and in research methods in the laboratory. The advances have assisted scientists in their study of bacterial gene complements and the roles of their gene products in the bacterial life cycle. Assignment of genes as essential to the bacterial cell nominated them as potential targets for antibacterial drugs and spurred attempts to exploit this information and convert it into drugs. At present, these efforts have met with minimal success. There are several possible reasons for these disappointing results including choice of targets and screen designs, compound libraries chosen for screens, and decreased commitment to antibacterial drug discovery by many large pharmaceutical companies. Structure-based approaches could become very effective in the future as methodologies continue to improve.

Array methodology singles out pathogenic bacteria

Bacteria are covered in sugars that facilitate the establishment of pathogenic or symbiotic relationships with other cells. Microarrays of carbohydrate-binding proteins now can provide quick snapshots of these sugar coats as they change during the bacterial life cycle and differ among bacterial strains.

Long-term survival during stationary phase: evolution and the GASP phenotype

The traditional view of the stationary phase of the bacterial life cycle, obtained using standard laboratory culture practices, although useful, might not always provide us with the complete picture. Here, the traditional three phases of the bacterial life cycle are expanded to include two additional phases: death phase and long-term stationary phase. In many natural environments, bacteria probably exist in conditions more akin to those of long-term stationary-phase cultures, in which the expression of a wide variety of stress-response genes and alternative metabolic pathways is essential for survival. Furthermore, stressful environments can result in selection for mutants that express the growth advantage in stationary phase (GASP) phenotype.

MODELING BACTERIA FATE AND TRANSPORT IN WATERSHEDS TO SUPPORT TMDLS

Fecal contamination of surface waters is a critical water-quality issue, leading to human illnesses and deaths. Total Maximum Daily Loads (TMDLs), which set pollutant limits, are being developed to address fecal bacteria impairments. Watershed models are widely used to support TMDLs, although their use for simulating in-stream fecal bacteria concentrations is somewhat rudimentary. This article provides an overview of fecal microorganism fate and transport within watersheds, describes current watershed models used to simulate microbial transport, and presents case studies demonstrating model use. Bacterial modeling capabilities and limitations for setting TMDL limits are described for two widely used watershed models (HSPF and SWAT) and for the load-duration method. Both HSPF and SWAT permit the user to discretize a watershed spatially and bacteria loads temporally. However, the options and flexibilities are limited. The models are also limited in their ability to describe bacterial life cycles and in their ability to adequately simulate bacteria concentrations during extreme climatic conditions. The load-duration method for developing TMDLs provides a good representation of overall water quality and needed water quality improvement, but intra-watershed contributions must be determined through supplemental sampling or through subsequent modeling that relates land use and hydrologic response to bacterial concentrations. Identified research needs include improved bacteria source characterization procedures, data to support such procedures, and modeling advances including better representation of bacteria life cycles, inclusion of more appropriate fate and transport processes, improved simulation of catastrophic conditions, and creation of a decision support tool to aid users in selecting an appropriate model or method for TMDL development.

Fitness Cost Due to Mutations in the 16S rRNA Associated with Spectinomycin Resistance in Chlamydia psittaci 6BC

The fitness cost of a resistance determinant is the primary parameter that determines its frequency in vivo. As a model for analysis of the impact of drug resistance mutations on the intracellular life cycle of Chlamydia spp., we studied the growth of four genetically defined spectinomycin-resistant (Spc(r)) clonal variants of Chlamydia psittaci 6BC isolated in the plaque assay. The development of each variant was monitored over 46 h postinfection in the absence of drug, either in pure culture or in 1:1 competition with the parent strain. Spc(r) mutations in the 16S rRNA gene at positions 1191 and 1193 were associated with a marked impairment of C.psittaci biological fitness, and the bacteria were severely out-competed by the wild-type parent. In contrast, mutations at position 1192 had minor effects on the bacterial life cycle, allowing the resistant isolates to compete more efficiently with the wild-type strain. Thus, mutations with a wide range of fitness costs can be selected in the plaque assay, providing a new strategy for prediction and monitoring of the emergence of antibiotic resistance in chlamydiae. So far, drug resistance has not been a serious threat for the treatment of chlamydial infections. Tetracycline is an effective antichlamydial drug that targets 16S rRNA. Attempts to isolate spontaneous tetracycline-resistant mutants of C. psittaci 6BC revealed a frequency <3 x 10(-9). We suggest that the rarity of genotypic antibiotic resistance among chlamydial clinical isolates reflects the deleterious effects of such mutations on the fitness of these obligate intracellular bacteria in the host.

Peptide deformylase inhibitors: a survey of the patent literature

New antibiotics with new mechanisms of action are desperately needed, as clinically significant bacterial pathogens have not only acquired resistance to nearly all existing antibiotics, but also increasingly exhibit multi-drug resistance. Peptide deformylase (PDF), a highly conserved metalloproteinase, is an essential enzyme in the bacterial life cycle. It catalyses the removal of the terminal formyl group from the N-terminal methionine residue of the nascent bacterial polypeptide chain. Thus, inhibition of PDF, by interfering with the bacterial protein maturation process, is providing a promising new and exciting target for antibiotic therapeutic intervention without interfering with eukaryotic metabolism. Antibiotics based on PDF inhibition have the potential to provide the much needed antibacterial activity against most of the major drug-resistant pathogens and could play a significant role in the treatment of community-acquired upper respiratory tract infections. This review covers all the patenting activity in this field of research up to, and including, March 2005, and will focus on the more significant patents and patent applications.

Bacterial Plurality as a General Mechanism Driving Persistence in Chronic Infections

Classical methods for the study of bacterial pathogens have proven to be inadequate to inform with respect to chronic infections including those associated with arthroplasties. Modern methods of analysis have demonstrated that bacterial growth patterns, ecology, and intra-species heterogeneity are more complex than were envisioned by early microbiologists. Cultural methods were developed to study acute, epidemic infections, but it is now recognized that the phenotype associated with these diseases represents only a minor aspect of the bacterial life cycle, which consists of planktonic, attachment, biofilm, and dispersal phases. Over 99% of bacteria in natural populations are found in biofilms which contain multiple ecological niches and numerous phenotypes. Unfortunately, the effort to develop antibiotics has been directed solely at the planktonic minority (associated with systemic illness) which explains our inability to eradicate chronic infections. In this study we establish a new rubric, bacterial plurality, for the understanding of bacterial ecology and evolution with respect to chronic infection. The fundamental tenets of bacterial plurality are that the bacteria within an infecting population display multiple phenotypes and possess multiple genotypes. Phenotypic plurality is embodied in the biofilm paradigm and genotypic plurality is embodied in the concepts of the supra-genome and the distributed genome hypothesis. It is now clear that bacterial diversity provides bacterial populations, as a whole, the ability to persist in the face of a multi-faceted host response.

The mathematical properties of the quasi-chemical model for microorganism growth–death kinetics in foods

Knowledge of the mathematical properties of the quasi-chemical model [Taub, Feeherry, Ross, Kustin, Doona, 2003. A quasi-chemical kinetics model for the growth and death of Staphylococcus aureus in intermediate moisture bread. J. Food Sci. 68 (8), 2530–2537], which is used to characterize and predict microbial growth–death kinetics in foods, is important for its applications in predictive microbiology. The model consists of a system of four ordinary differential equations (ODEs), which govern the temporal dependence of the bacterial life cycle (the lag, exponential growth, stationary, and death phases, respectively). The ODE system derives from a hypothetical four-step reaction scheme that postulates the activity of a critical intermediate as an antagonist to growth (perhaps through a quorum sensing biomechanism). The general behavior of the solutions to the ODEs is illustrated by several examples. In instances when explicit mathematical solutions to these ODEs are not obtainable, mathematical approximations are used to find solutions that are helpful in evaluating growth in the early stages and again near the end of the process. Useful solutions for the ODE system are also obtained in the case where the rate of antagonist formation is small. The examples and the approximate solutions provide guidance in the parameter estimation that must be done when fitting the model to data. The general behavior of the solutions is illustrated by examples, and the MATLAB programs with worked examples are included in the appendices for use by predictive microbiologists for data collected independently.