MicrobiologyBytes

How bacteria grow and divide while retaining a defined shape is a fundamental question in microbiology, but technological advances are now driving a new understanding of how the shape-maintaining bacterial peptidoglycan sacculus grows. This review highlights the relationship between peptidoglycan synthesis complexes and cytoskeletal elements, as well as recent evidence that peptidoglycan growth is regulated from outside the sacculus in Gram-negative bacteria. It also discusses how growth of the sacculus is sensitive to mechanical force and nutritional status, and describe the roles of peptidoglycan hydrolases in generating cell shape and of D-amino acids in sacculus remodelling.

From the regulation of peptidoglycan synthesis to bacterial growth and morphology. (2012) Nature Reviews Microbiology 10: 123-136 doi:10.1038/nrmicro2677

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What would happen if all the leaves fell off the trees and did not rot? We’d be buried under them and all plants would run out of nutrients and die, then we would starve. So the seemingly non-sexy buisness of rotting is rather important when it comes to element and nutrient cycles.

In ecological studies, leaf litter degradation is often estimated by measuring parameters such as soil respiration, litter mass loss or the activities of specific microbial enzymes in soil extracts. In microbiology, the degradation of plant-derived compounds such as lignocellulose has been studied using a few microbial model species and has recently led to the sequencing of the genomes of different saprotrophic fungal species which use different strategies to degrade plant material, thus revealing the full enzymatic machinery implicated in this process. Under natural conditions, litter degradation is generally carried out by consortia of species that either act simultaneously or replace one another on a common piece of plant debris in a sometimes predictable manner and not by a single microbial species. It can therefore be anticipated that the molecular machinery deployed to completely mineralize litter in the field is far more complex and diverse than the machinery observed in a single microbial genome. In addition, it is likely that the diversity of this machinery is partly controlled by litter chemistry and complexity and therefore by plant community composition.

By allowing access to the genome contents of the different microorganisms present in a common environment (metagenomics) or to the set of genes they express (metatranscriptomics), environmental genomics offers a novel opportunity to decipher at the molecular level, complex ecological processes such as plant organic matter degradation, thus bridging the gap between global field measurements and targeted genomic approaches.

Metatranscriptomics Reveals the Diversity of Genes Expressed by Eukaryotes in Forest Soils. (2012) PLoS ONE 7(1): e28967. doi:10.1371/journal.pone.0028967
Eukaryotic organisms play essential roles in the biology and fertility of soils. For example the micro and mesofauna contribute to the fragmentation and homogenization of plant organic matter, while its hydrolysis is primarily performed by the fungi. To get a global picture of the activities carried out by soil eukaryotes we sequenced 2×10,000 cDNAs synthesized from polyadenylated mRNA directly extracted from soils sampled in beech (Fagus sylvatica) and spruce (Picea abies) forests. Taxonomic affiliation of both cDNAs and 18S rRNA sequences showed a dominance of sequences from fungi (up to 60%) and metazoans while protists represented less than 12% of the 18S rRNA sequences. Sixty percent of cDNA sequences from beech forest soil and 52% from spruce forest soil had no homologs in the GenBank/EMBL/DDJB protein database. A Gene Ontology term was attributed to 39% and 31.5% of the spruce and beech soil sequences respectively. Altogether 2076 sequences were putative homologs to different enzyme classes participating to 129 KEGG pathways among which several were implicated in the utilisation of soil nutrients such as nitrogen (ammonium, amino acids, oligopeptides), sugars, phosphates and sulfate. Specific annotation of plant cell wall degrading enzymes identified enzymes active on major polymers (cellulose, hemicelluloses, pectin, lignin) and glycoside hydrolases represented 0.5% (beech soil)–0.8% (spruce soil) of the cDNAs. Other sequences coding enzymes active on organic matter (extracellular proteases, lipases, a phytase, P450 monooxygenases) were identified, thus underlining the biotechnological potential of eukaryotic metatranscriptomes. The phylogenetic affiliation of 12 full-length carbohydrate active enzymes showed that most of them were distantly related to sequences from known fungi. For example, a putative GH45 endocellulase was closely associated to molluscan sequences, while a GH7 cellobiohydrolase was closest to crustacean sequences, thus suggesting a potentially significant contribution of non-fungal eukaryotes in the actual hydrolysis of soil organic matter.

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Iron is a highly abundant metal on earth and is vital for virtually all organisms. Despite its abundance, iron deficiency is the most prevalent nutrition disorder worldwide. It mostly affects infants, young children and women in developing countries. Iron deficiency has major health consequences such as infection, poor pregnancy outcome, and impaired physical and cognitive development. Several trials have shown that iron deficiency can be effectively controlled by both iron supplementation and fortification programmes. However, safety of iron supplementation has been questioned and there is evidence suggesting that untargeted oral iron supplementation in regions with high prevalence of malaria transmission and infectious diseases can cause an increase in infections, hospital admission and mortality in young children. This might be at least partly ascribed to iron also being an essential requirement for the growth of most bacterial species. Importantly, iron availability is frequently involved in the expression of virulence-associated properties in pathogenic bacteria:

Iron Availability Increases the Pathogenic Potential of Salmonella Typhimurium and Other Enteric Pathogens at the Intestinal Epithelial Interface. (2012) PLoS ONE 7(1): e29968. doi:10.1371/journal.pone.0029968
Recent trials have questioned the safety of untargeted oral iron supplementation in developing regions. Excess of luminal iron could select for enteric pathogens at the expense of beneficial commensals in the human gut microflora, thereby increasing the incidence of infectious diseases. The objective of the current study was to determine the effect of high iron availability on virulence traits of prevalent enteric pathogens at the host-microbe interface. A panel of enteric bacteria was cultured under iron-limiting conditions and in the presence of increasing concentrations of ferric citrate to assess the effect on bacterial growth, epithelial adhesion, invasion, translocation and epithelial damage in vitro. Translocation and epithelial integrity experiments were performed using a transwell system in which Caco-2 cells were allowed to differentiate to a tight epithelial monolayer mimicking the intestinal epithelial barrier. Growth of Salmonella typhimurium and other enteric pathogens was increased in response to iron. Adhesion of S. typhimurium to epithelial cells markedly increased when these bacteria were pre-incubated with increasing iron concentration), whereas this was not the case for the non-pathogenic Lactobacillus plantarum. Cellular invasion and epithelial translocation of S. typhimurium followed the trend of increased adhesion. Epithelial damage was increased upon incubation with S. typhimurium or Citrobacter freundii that were pre-incubated under iron-rich conditions. In conclusion, our data fit with the consensus that oral iron supplementation is not without risk as iron could, in addition to inducing pathogenic overgrowth, also increase the virulence of prevalent enteric pathogens.

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Staphylococcus aureus is responsible for the vast majority of bacterial skin infections in humans. The propensity for S. aureus to infect skin involves a balance between cutaneous immune defense mechanisms and virulence factors of the pathogen. The tissue architecture of the skin is different from other epithelia especially since it possesses a corneal layer, which is an important barrier that protects against the pathogenic microorganisms in the environment. The skin surface, epidermis, and dermis all contribute to host defense against S. aureus. Conversely, S. aureus utilizes various mechanisms to evade these host defenses to promote colonization and infection of the skin.

This review focuses on host-pathogen interactions at the skin interface during the pathogenesis of S. aureus colonization and infection.

Host-pathogen interactions between the skin and Staphylococcus aureus. Curr Opin Microbiol. 01 Dec 2011

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Regulons are the basic units of cellular response systems in bacterial cells, and represent a most basic concept in bacterial studies. A bacterial regulon is a group of operons that are transcriptionally co-regulated by the same regulatory machinery, consisting of trans regulators (transcription factors or simply TFs) and cis regulatory binding elements in the promoters of the operons they regulate. Operationally, a regulon contains operons regulated by one same transcription factor. Since the term regulon was first proposed in 1964, 173 regulons have been fully or partially identified in E. coli K12 and many more in other bacteria e.g. B. subtilis.

Loosely speaking, regulons can be categorized into two classes: local and global regulons, with the former corresponding to regulons consisting of only a few component operons and the latter having a relatively large number of operons. While the functionalities of the known regulons have been well studied, very little is known about how regulons are organized in a bacterial genome.

This paper examines the organizational principles of regulons in bacterial genomes, looking at E. coli K12 and Bacillus subtilis. The key findings are (1) operons of each regulon tend to form a few closely located clusters along with genome; (2) TFs are under stronger evolutionary constraints than their TGs; and (3) the global arrangement of the component operons of all the (known) regulons in a genome tend to minimize a simple scoring function.

Genomic Arrangement of Regulons in Bacterial Genomes. (2012) PLoS ONE 7(1): e29496. doi:10.1371/journal.pone.0029496
Regulons, as groups of transcriptionally co-regulated operons, are the basic units of cellular response systems in bacterial cells. While the concept has been long and widely used in bacterial studies since it was first proposed in 1964, very little is known about how its component operons are arranged in a bacterial genome. We present a computational study to elucidate of the organizational principles of regulons in a bacterial genome, based on the experimentally validated regulons of E. coli and B. subtilis. Our results indicate that (1) genomic locations of transcriptional factors (TFs) are under stronger evolutionary constraints than those of the operons they regulate so changing a TF’s genomic location will have larger impact to the bacterium than changing the genomic position of any of its target operons; (2) operons of regulons are generally not uniformly distributed in the genome but tend to form a few closely located clusters, which generally consist of genes working in the same metabolic pathways; and (3) the global arrangement of the component operons of all the regulons in a genome tends to minimize a simple scoring function, indicating that the global arrangement of regulons follows simple organizational principles. Tweet

Traditional approaches to phage therapy rely on the ability of viruses to kill their bacterial prey. However, the narrow host range or most bacteriophages and the ability of bacteria to become resistant to infection mean that in practice, using phage to simply replace antibiotics is not feasible. We need smarter approaches, which is where a recent paper comes in. Using phages to engineer sensitivity to antibiotics is a promising approach, but whether this proof-of-principle experiment ever makes it to the clinic is another matter.

Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes. (2011)Appl. Environ. Microbiol. 23 Nov 2011 doi: 10.1128/AEM.05741-11
Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that will resensitize pathogens to approved antibiotics therefore holds key advantages. We present a proof-of-principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages encoding the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene’s sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage’s ability to kill pathogens in the infected host, but instead, to deliver genetic constructs into the bacteria, and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phages will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.

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The structural complexities of bacteria are becoming increasingly apparent. Gram-negative bacteria can be divided into several subcellular compartments. There are two aqueous compartments called the cytoplasm and the periplasm. The cytoplasm is enclosed by a phospholipid bilayer called the inner membrane (IM), which is itself surrounded by an asymmetric bilayer called the outer membrane (OM). The periplasm lies in the space between the IM and OM, and is home to the peptidoglycan cell wall (CW). Present throughout these compartments are proteins with diverse and important biological functions. Some of these proteins are membrane-embedded and allow the transfer of molecules between compartments. Others are soluble enzymes involved in metabolic reactions. Much work has been devoted toward understanding how each of these compartments is formed and maintained. This review focuses on a particular aspect of OM biogenesis, namely the assembly of integral outer membrane proteins (OMPs).

Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria. Curr Opin Microbiol. 03 Jan 2012
The outer membrane (OM) of Gram-negative bacteria is an essential organelle that serves as a selective permeability barrier by keeping toxic compounds out of the cell while allowing vital nutrients in. How the OM and its constituent lipid and protein components are assembled remains an area of active research. In this review, we describe our current understanding of how outer membrane proteins (OMPs) are delivered to and then assembled in the OM of the model Gram-negative organism Escherichia coli.

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Cystic fibrosis is caused by mutations in the CFTR gene leading to a disrupted chloride channel. It is well established that the greatest contributor to patient morbidity and mortality is chronic lung disease, caused by a constant cycle of infection and inflammation throughout the patient’s life. The CFTR mutation leads to defective regulation of chloride and sodium, resulting in increased water absorption, depletion of airway surface liquid and dehydrated mucous. Consequently, the purulent sputum and mucus plugs together with an ineffective inflammatory response, all contribute to the chronic infections that are central to CF lung disease. From early childhood, CF patients experience recurrent pulmonary infections from a range of pathogens.

In spite of intensive antibiotic therapy, certain organisms persist, leading to pulmonary exacerbations, hospitalizations and patient death. These include Pseudomonas aeruginosa, Burkholderia cepacia complex (Bcc) and Achromobacter xylosoxidans, with Bcc being the most problematic. It was recently demonstrated that chronic colonisation by Bcc resulted in a greater lung function decline than by the other two pathogens. CF patients are also susceptible to colonisation by other pathogens, including Staphylococcus aureus (both methicillin-resistant and sensitive), genus Pandoraea, Stenotrophomonas maltophilia and non-tuberculous Mycobacteria, although the role of these latter four pathogens in CF lung disease is unclear. Furthermore, the identification of high levels of anaerobic organisms in CF sputum has added to the complex microbial population in the CF lung. These CF-associated anaerobes were not susceptible to antibiotics with known efficacy against anaerobes and the clinical significance of anaerobes in CF is not yet fully understood.

Bacterial host interactions in cystic fibrosis. Curr Opin Microbiol. Dec 1 2011
Chronic infection is a hallmark of cystic fibrosis (CF) and the main contributor to morbidity. Microbial infection in CF is complex, due to the number of different species that colonise the CF lung. Their colonisation is facilitated by a host response that is impaired or compromised by highly viscous mucous, zones of hypoxia and the lack of the cystic fibrosis transmembrane regulator (CFTR). Successful dominant CF pathogens combine an effective arsenal to establish infection and counter-attack the host response, together with an ability to adapt readily to an unfavourable environment. Hypermutability is common among CF pathogens facilitating adaptation and as the host response persists, progressive destruction of the normal architecture of lung tissue ensues with catastrophic consequences for the host.

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DNA fragments encoding enzymes, transcriptional regulators and virulence factors are fluxing through bacterial taxonomic walls by horizontal gene transfer. These elements often endow environmental and clinical strains of bacteria with new properties, including an enhanced virulence. Lateral genetic exchange, particularly of drug tolerance genes has been recognized for a long time; however, our understanding of this phenomenon is limited. Ontology and phylogeny of laterally transferred genetic elements are difficult to investigate, let alone the predictions of their insertion sites in hosts chromosomes.

An outbreak of the lethal Escherichia coli in Europe in 2011 highlighted the shortcoming of our knowledge on the basic principles of evolutionary trends of new pathogens. The outbreak first occurred in Germany in May 2011 where a rare enterohemorrhagic strain Escherichia coli O104:H4 caused haemolytic-uremic syndrome. The infection spread fast through many other European countries and sickened thousands of people. The level of lethality associated with the production of Shiga toxin by the strain and its resistance against many antibiotics was significant. A number of isolates from this outbreak have been sequenced and annotated. Based on the unique combination of genomic features these strains were suggested to represent a new pathotype Entero-Aggregative-Haemorrhagic E. coli (EAHEC).

Mainstreams of Horizontal Gene Exchange in Enterobacteria: Consideration of the Outbreak of Enterohemorrhagic E. coli O104:H4 in Germany in 2011. (2011) PLoS ONE 6(10): e25702. doi:10.1371/journal.pone.0025702
Escherichia coli O104:H4 caused a severe outbreak in Europe in 2011. The strain TY-2482 sequenced from this outbreak allowed the discovery of its closest relatives but failed to resolve ways in which it originated and evolved. On account of the previous statement, may we expect similar upcoming outbreaks to occur recurrently or spontaneously in the future? The inability to answer these questions shows limitations of the current comparative and evolutionary genomics methods. The study revealed oscillations of gene exchange in enterobacteria, which originated from marine γ-Proteobacteria. These mobile genetic elements have become recombination hotspots and effective ‘vehicles’ ensuring a wide distribution of successful combinations of fitness and virulence genes among enterobacteria. Two remarkable peculiarities of the strain TY-2482 and its relatives were observed: i) retaining the genetic primitiveness by these strains as they somehow avoided the main fluxes of horizontal gene transfer which effectively penetrated other enetrobacteria; ii) acquisition of antibiotic resistance genes in a plasmid genomic island of β-Proteobacteria origin which ontologically is unrelated to the predominant genomic islands of enterobacteria. Oscillations of horizontal gene exchange activity were reported which result from a counterbalance between the acquired resistance of bacteria towards existing mobile vectors and the generation of new vectors in the environmental microflora. We hypothesized that TY-2482 may originate from a genetically primitive lineage of E. coli that has evolved in confined geographical areas and brought by human migration or cattle trade onto an intersection of several independent streams of horizontal gene exchange. Development of a system for monitoring the new and most active gene exchange events was proposed. Tweet