Search

The recent rapid expansion in the DNA and protein databases, arising from large-scale genomic and metagenomic sequence projects, has forced significant development in the field of phylogenetics: the study of the evolutionary relatedness of the planet’s inhabitants. Advances in phylogenetic analysis have greatly transformed our view of the landscape of evolutionary biology, transcending the view of the tree of life that has shaped evolutionary theory since Darwinian times. Indeed, modern phylogenetic analysis no longer focuses on the restricted Darwinian–Mendelian model of vertical gene transfer, but must also consider the significant degree of lateral gene transfer, which connects and shapes almost all living things. Herein, I review the major tree-building methods, their strengths, weaknesses and future prospects.

This review focuses on the most recent advances in the application of probiotics as potential therapeutics for the developing world, from the treatment of chronic and acute enteric infections and their associated diarrheal complexes to the development of designer probiotics for controlling HIV and as novel mucosal vaccine delivery vehicles.

Canine parvovirus (CPV) and canine coronavirus (CCoV) are considered the main pathogens responsible for acute gastroenteritis in dogs. From a collection of 250 samples, seven CPV strains and three CCoV strains were identified in symptomatic Irish dogs. Samples were screened for the viruses using polymerase chain reaction (PCR) and typed via DNA sequence analysis. Three CPV strains were characterized as CPV-2a, while four others were characterized as CPV-2b. To date, CPV-2c remains unreported in Ireland. Two CCoV strains were characterized as CCoV-II and one as CCoV-I. In the case of one sample, PH4/09/Ire, a mixed infection with CPV and CCoV was detected.

As sequence data continues to be generated at a logarithmic rate our dependence on effective in silico gene prediction methods is also increasing. Herein, I review the current state of eukaryote gene prediction methods; their strengths, weaknesses and future directions.

Publisher Summary Many potentially beneficial probiotics often prove to be physiologically fragile, a significant limitation in clinical applications. Improving probiotic stress tolerance and the ability to grow and survive in foods prior to ingestion and subsequently within the animal host is an important clinical goal. The pathobiotechnology concept seeks to attain this goal, ultimately leading to the development of improved probiotic strains. A primary focus of this approach involves equipping probiotic bacteria with the genetic elements necessary to overcome the many stresses encountered during the probiotic life cycle (both external and internal to the host) as well as enabling probiotics to better deal with invading pathogens. Engineered probiotics thus have the potential to alleviate the symptoms of chronic gastrointestinal disorders and associated sequelae, to fight infection, modulate the immune system, and act as delivery vehicles for bioactive molecules. Notwithstanding these impressive health benefits, probiotic research has really only begun to achieve scientific credibility over the last decade. However, the increasing emergence of antibiotic resistance, coupled with a significant decline in production of new antibacterials, means that probiotics are finally coming of age, representing a real alternative to traditional drug-based therapies.

As the protein databases continue to expand at an exponential rate, fed by daily uploads from multiple large scale genomic and metagenomic projects, the problem of assigning a function to each new protein has become the focus of significant research interest in recent times. Herein, we review the most recent advances in the field of automated function prediction (AFP). We begin by defining what is meant by biological “function” and the means of describing such functions using standardised machine readable ontologies. We then focus on the various function-prediction programs available, both sequence and structure based, and outline their associated strengths and weaknesses. Finally, we conclude with a brief overview of the future challenges and outstanding questions in the field, which still remain unanswered.

While seven penicillin-binding proteins (PBPs) or PBP-like proteins have been identified either by radiolabelled penicillin binding studies or genomic analysis, only PBP3 has been considered of interest for Beta-lactams activity againstListeria monocytogenes. Herein we reveal that both PBP4 and Lmo0441 (a PBP-like protein) play a direct role in cephalosporin activity inL. monocytogeneswhile PBP4 additionally has a protective affect against both penicillin and carbapenem.

Bovine group A rotavirus is one of the main causes of neonatal diarrhoea in calves. This study examined the different G and P genotypes circulating in the bovine population, from 2002-2009, in the south of Ireland. Rotavirus positive bovine faecal samples (n=332) were collected from the Cork Regional Veterinary Laboratory, between 2002 and 2009 and subjected to RNA extraction, PAGE analysis, and G and P genotyping. Genotyping analysis identified G6, G10, P[5], and P[11] to be the predominant G and P genotypes in the present study, with G6 rotavirus responsible for 70-80% of rotavirus infections. The highest combination of G and P types found was G6 P[5], followed by G6 P[5+11] mixed infection. The prevalence of G6 and G10 has shifted over the years, with an increase in the amount of G10 P[11] being detected. Novel combinations (G6+G10P[11], G6+G10P[5+11] and G10P[5+11]) were also detected for the first time. In addition to this, sequence analysis of the VP7 RT-PCR amplicons has revealed that Irish G6 strains are falling within three different lineages, III-V. During this study, two samples, initially genotyped as G8P[11] were identified through sequence analysis as being true G6, lineage III with a high nucleotide identity to Hun4, a G6 human sample from Hungary. The increase in novel G and P type combinations, as well as changes seen in G6 samples could have an impact on rotavirus vaccination programmes, as the current vaccine available may not offer protection against all of these circulating types.

The food-borne pathogenic bacterium Listeria monocytogenes has the potential to adapt to an array of suboptimal growth environments encountered within the host. The pathogen is relatively bile tolerant and has the capacity to survive and grow within both the small intestine and the gallbladder in murine models of oral infection. We have previously demonstrated a role for the principal carnitine transport system of L. monocytogenes (OpuC) in gastrointestinal survival of the pathogen (R. Sleator, J. Wouters, C. G. M. Gahan, T. Abee, and C. Hill, Appl. Environ. Microbiol. 67:2692-2698, 2001). However, the mechanisms by which OpuC, or indeed carnitine, protects the pathogen in this environment are unclear. In the current study, systematic analysis of strains with mutations in osmolyte transporters revealed a role for OpuC in resisting the acute toxicity of bile, with a minor role also played by BetL, a secondary betaine uptake system which also exhibits a low affinity for carnitine. In addition, the toxic effects of bile on wild-type L. monocytogenes cells were ameliorated when carnitine (but not betaine) was added to the medium. lux -promoter fusions to the promoters of the genes encoding the principal osmolyte uptake systems Gbu, BetL, and OpuC and the known bile tolerance system BilE were constructed. Promoter activity for all systems was significantly induced in the presence of bile, with the opuC and bilE promoters exhibiting the highest levels of bile-dependent expression in vitro and the betL and bilE promoters showing the highest expression levels in the intestines of orally inoculated mice. A direct comparison of all osmolyte transporter mutants in a murine oral infection model confirmed a major role for OpuC in intestinal persistence and systemic invasion and a minor role for the BetL transporter in fecal carriage. This study therefore demonstrates a previously unrecognized function for osmolyte uptake systems in bile tolerance in L. monocytogenes .

Carnitine has beneficial vitamin-like qualities and functions as a compatible solute. The presence of L-carnitine in infant formula significantly promoted the growth of Listeria monocytogenes at 7 degrees C, whereas disruption of the genes for carnitine and glycine betaine uptake compromised growth of the L. monocytogenes mutant in formula containing carnitine that was stored at 7 degrees C. Thus, addition of carnitine to baby formula may pose a potential food safety risk.

Background Probiotics such as bifidobacteria have been shown to maintain a healthy intestinal microbial balance and help protect against infections. However, despite these benefits, bifidobacteria still remain poorly understood at the biochemical, physiological and especially the genetic level. Herein we describe, for the first time, the development of a non-invasive luciferase-based reporter system for real-time tracking of Bifidobacterium species in vivo. Results The reporter vector pLuxMC1 is based on the recently described theta-type plasmid pBC1 from B. catenatulatum [1] and the luxABCDE operon from pPL2lux [2]. Derivatives of pLuxMC1, harbouring a bifidobacterial promoter (pLuxMC2) as well as a synthetically derived promoter (pLuxMC3) [3] placed upstream of luxABCDE, were constructed and found to stably replicate in B. breve UCC2003. The subsequent analysis of these strains allowed us to assess the functionality of pLuxMC1 both in vitro and in vivo. Conclusion Our results demonstrate the potential of pLuxMC1 as a real-time, non-invasive reporter system for Bifidobacterium. It has also allowed us, for the first time, to track the colonisation potential and persistence of this probiotic species in real time. An interesting and significant outcome of the study is the identification of the caecum as a niche environment for B. breve UCC2003 within the mouse gastrointestinal tract (GI) tract.

BackgroundThe majority of commensal gastrointestinal bacteria used as probiotics are highly adapted to the specialised environment of the large bowel. However, unlike pathogenic bacteria; they are often inadequately equipped to endure the physicochemical stresses of gastrointestinal (GI) delivery in the host. Herein we outline a patho-biotechnology strategy to improve gastric delivery and host adaptation of a probiotic strainBifidobacterium breveUCC2003 and the generally regarded as safe (GRAS) organismLactococcus lactisNZ9000.ResultsIn vitrobile tolerance of both strains was significantly enhanced (P< 0.001), following heterologous expression of theListeria monocytogenesbile resistance mechanism BilE. Strains harbouringbilEwere also recovered at significantly higher levels (P< 0.001), than control strains from the faeces and intestines of mice (n= 5), following oral inoculation. Furthermore, aB. brevestrain expressingbilEdemonstrated increased efficacy relative to the wild-type strain in reducing oralL. monocytogenesinfection in mice.ConclusionCollectively the data indicates that bile tolerance can be enhanced inBifidobacteriumandLactococcusspecies through rational genetic manipulation and that this can significantly improve delivery to and colonisation of the GI tract.

High-pressure processing (HPP) is a nonthermal process capable of inactivating and eliminating pathogenic and food spoilage microorganisms. This novel technology has enormous potential in the food industry, controlling food spoilage, improving food safety and extending product shelf life while retaining the characteristics of fresh, preservative-free, minimally processed foods. As with other food processing methods, such as thermal processing, HPP has somewhat limited applications as it cannot be universally applied to all food types, such as some dairy and animal products and shelf-stable low-acid foods. Herein, we discuss the effects of high-pressure processing on microbial food safety and, to a lesser degree, food quality.

Given the increasing commercial and clinical relevance of probiotic cultures, improving their stress tolerance profile and ability to overcome the physio-chemical defences of the host is an important biological goal. Pathogenic bacteria have evolved sophisticated strategies to overcome host defences, interact with the immune system and interfere with essential host systems. We coin the term ‘patho-biotechnology’ to describe the exploitation of these valuable traits in biotechnology and biomedicine. This approach shows promise for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications as well as the development of novel vaccine and drug delivery platforms.

While much information is available concerning the health promoting benefits associated with dietary reformulations (e.g. replacing sugar with aspartame and salt (NaCl) with KCl), to reduce the incidence of obesity and heart disease, potential food safety risks arising from such reformulations have received considerably less attention. Reformulation inevitably changes the intrinsic physico-chemical properties of the food, which may in turn support the growth of food-borne pathogens and ultimately their ability to cause disease. Thus, a better understanding of the microbiological food safety issues associated with product reformulation for increased health are required.

In addition to the severe invasive systemic disease of listeriosis, recent evidence suggests that the Gram-positive intracellular bacterial pathogen Listeria monocytogenes is also a causative agent of febrile gastroenteritis. We examined the listerial response to stresses normally encountered in the upper small intestine and demonstrate that osmotic stress appears to be at the top of the hierarchy of stress responses during gastrointestinal residence. Furthermore, we suggest that the increased osmolarity of the gastrointestinal lumen may be interpreted as an environmental cue signalling gut entry and that the underlying genetic element governing this response is the alternative stress sigma factor sigma(B).

Given the increasing commercial and clinical relevance of probiotic cultures, improving the technological robustness of what are often process-sensitive cultures is an important biological goal. The nisin-controlled expression system was used to direct the heterologous expression of the listerial betaine uptake system BetL in the probiotic strain Lactobacillus salivarius UCC118. Following nisin induction, strains expressing betL exhibited a significant increase in resistance to several stresses, including elevated osmo-, cryo-, baro-, and chill tolerance, as well as increased resistance to spray- and freeze-drying. The ability to confer additional stress tolerance on a probiotic culture may be an important step in delivering viable cultures for maximal efficacy.

Pathogenic bacteria have evolved sophisticated strategies to overcome host defences, to interact with the immune system and to interfere with essential host systems. We coin the term ‘patho-biotechnology’ to describe the exploitation of these valuable traits in biotechnology, medicine and food. This approach shows promise for the development of novel vaccine and drug delivery systems, as well as for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications. The genetic tractability of Listeria monocytogenes, the availability of the complete genome sequence of this intracellular pathogen, its ability to cope with stress, and its ability to traverse the gastrointestinal tract and induce a strong cellular immune response make L. monocytogenes an ideal model organism for demonstrating the patho-biotechnology concept.

In silico analysis of the Listeria monocytogenes genome revealed lmo0292 , a gene predicted to encode a HtrA-like serine protease. A stable insertion mutant was constructed, revealing a requirement for htrA in the listerial response to heat, acid, and penicillin stress. Transcriptional analysis revealed that htrA is not induced in response to heat shock but is induced in response to low pH and penicillin G stress. Furthermore, htrA expression was shown to be dependent upon the LisRK two-component sensor-kinase, a system known to respond to changes in integrity of the cell envelope. In addition, we demonstrated that a second in-frame start codon, upstream of that previously annotated for L. monocytogenes htrA , incorporating a putative signal sequence appears to influence virulence potential. Finally, a significant virulence defect was observed for the htrA mutant, indicating that this gene is required for full virulence in mice. Our findings suggest that L. monocytogenes lmo0292 encodes an HtrA-like serine protease that is not part of the classical heat shock response but is involved in stress responses and virulence.

Understanding how pathogenic bacteria sense and respond to environmental signals, including those involved in triggering virulence gene expression, is a fundamental biological goal. It is now known that the two-component regulatory system LisRK has a novel role in osmosensing and osmoregulation in the intracellular foodborne pathogen Listeria monocytogenes. Furthermore, htrA, a gene linked to osmotolerance and virulence potential in L. monocytogenes, is now known to be under the transcriptional control of LisRK.

The ability to colonize the gall bladder has recently been shown to be an important feature of virulent Listeria monocytogenes (J. Hardy, K. P. Francis, M. DeBoer, P. Chu, K. Gibbs, C. H. Contag. Science 303: 851-853, 2004). We suggest that the cytotoxic effects of bile may be increased upon release from the gall bladder into the upper small intestine, and report the identification of a novel bile exclusion system which plays an essential role in intestinal colonization and virulence of L. monocytogenes. In silico analysis of the L. monocytogenes EGDe genome revealed a two-gene operon (formerly opuB) exhibiting significant sequence similarity to members of the betaine carnitine choline transporter (BCCT) family. The operon, herein designated bilE (bile Exclusion) is preceded by consensus sigmaA- and sigmaB-dependent promoter-binding sites and is transcriptionally upregulated at elevated osmolarities and reduced temperatures (stresses known to induce sigB). Furthermore, a significant reduction in the level of bilE transcription was observed in the absence of sigmaB. In addition, we demonstrate an important role for PrfA, the master regulator of virulence potential in L. monocytogenes, in coordinating bilE expression. Computational structural analysis suggests that, rather than functioning as a compatible solute uptake system as was previously believed, BilE is more likely to be an exclusion system, a conclusion substantiated by radiolabelled bile accumulation studies. In addition, functionally inactivating BilE resulted in a five-log reduction in the ability of the bacterium to tolerate lethal concentrations of bovine bile (oxgall) and also significantly increased sensitivity to physiological concentrations of human bile, a phenotype which translates to a significant reduction in virulence potential when administered to a murine model by the oral route. Thus, this novel bile exclusion locus bilE, coordinately regulated by sigmaB and PrfA, represents a new and important virulence factor in L. monocytogenes.

Listeria monocytogenes must resist the deleterious actions of bile in order to infect and subsequently colonize the human gastrointestinal tract. The molecular mechanisms used by the bacterium to resist bile and the influence of bile on pathogenesis are as yet largely unexplored. This study describes the analysis of three genes— bsh , pva , and btlB —previously annotated as bile-associated loci in the sequenced L. monocytogenes EGDe genome (lmo2067, lmo0446, and lmo0754, respectively). Analysis of deletion mutants revealed a role for all three genes in resisting the acute toxicity of bile and bile salts, particularly glycoconjugated bile salts at low pH. Mutants were unaffected in the other stress responses examined (acid, salt, and detergents). Bile hydrolysis assays demonstrate that L. monocytogenes possesses only one bile salt hydrolase gene, namely, bsh . Transcriptional analyses and activity assays revealed that, although it is regulated by both PrfA and σ B , the latter appears to play the greater role in modulating bsh expression. In addition to being incapable of bile hydrolysis, a sigB mutant was shown to be exquisitely sensitive to bile salts. Furthermore, increased expression of sigB was detected under anaerobic conditions and during murine infection. A gene previously annotated as a possible penicillin V amidase ( pva ) or bile salt hydrolase was shown to be required for resistance to penicillin V but not penicillin G but did not demonstrate a role in bile hydrolysis. Finally, animal (murine) studies revealed an important role for both bsh and btlB in the intestinal persistence of L. monocytogenes .

Recent work from the Leigh lab, published in Science,1 describes the design, synthesis and operation of an artificial small-molecule capable of synthesizing peptides in a sequence-specific manner. The design of this artificial molecular machine was, in the author’s own words, “inspired by the ribosome”2; with several elements analogous to ribosomal protein synthesis (Fig. 1). The tRNA’s of conventional translation are substituted by amino acids attached to a track (or axle) by weak phenolic ester linkages. The sequence and spacing of the axle-tethered amino acids enables sequence specific peptide synthesis (analogous to the mRNA template in nature). Using copper ions to direct the process, attachment of the artificial molecular machine to the axle is mediated by a macrocycle—a large nanometer-sized molecular ring that straddles the axle and moves along its axis in a manner reminiscent of the ribosome subunits clamping to and moving along the mRNA strand. Attached to the ring is a reactive arm (a cysteine derivative bearing a trityl (Trt)-protected thiol group) which detaches the amino acids from the axle, passing them to the elongation site (a tert-butoxycarbonyl carbamate (Boc)—protected amine at the end of a glycylglycine residue), where the resulting peptide oligomer is synthesized in a single specific sequence, through native chemical elongation.3 Figure 1. Schematic representation of the synthetic ribosome; illustrating the macrocycle with attached reactive arm and the axle with attached amino acids The synthesis reaction is initiated by acid-catalyzed cleavage of the Boc and trityl protecting groups; activating the molecular machine, which then operates at 60°C under microwave heating in the presence of a reducing agent (which cleaves any disulphide bonds formed through thiol oxidation). Once the thiolate residue of the catalytic cysteine group is deprotected, it undergoes a transacylation reaction with the first amino acid phenolic ester that blocks the ring as it slides along the axle. The subsequently formed phenylalanine thioester then transfers the amino acid to the glycylglycine amine group. This sequence simultaneously transfers the amino acid to the end of the growing peptide and regenerates the catalytic thiolate group, ready for cleavage and transfer of further amino acids. Once the covalent bond linking the amino acid to the axle is broken, the macrocycle is free to move further along the axle until it reaches the next amino acid in the sequence, and the entire process is repeated. When the last amino acid on the axle is cleaved the macrocycle detaches with the newly formed, full length peptide attached. Unlike ribosomal translation, the artificial molecular machine synthesizes the peptide in the opposite direction i.e., from the C- to the N-terminus. Proof that the process works was obtained using a combination of HPLC (which showed that none of the starting material remained), 1H NMR and mass spectrometry—which identified the completely deacylated thread (or axle) and the macrocycle bearing the synthesized hexapeptide (Piv)AlaLeuPheGlyGlyCys [Piv,COC(CH3)3]. The peptide sequence was confirmed by tandem mass spectrometry (MS/MS). Although only about one-tenth the size of nature’s ribosomes,4 Leighs’s peptide synthesizer is capable of producing peptides in a scale of tens of milligrams (corresponding to parallel synthesis by ~108 molecular machines). However, taking ~12 h to make each peptide bond, the synthetic system remains a “primitive analog of the ribosome”2 which is capable of synthesizing ~15 to 20 amide bonds per second. Furthermore, the rotaxane destroys the code it reads, and can produce only relatively short peptide strands. Nonetheless, the current study provides a valuable proof of concept; a first generation prototype which is likely to be improved upon. One suggested upgrade is to replace the preloaded axle with amino acid binding sites (as opposed to the amino acids themselves). Using this approach, once the amino acids have been picked in the first round, the axle could reload (by binding fresh amino acids from the bathing solution), thereby reversing the loss of sequence information on the strand. Leigh’s rotaxane is the latest in a growing list of artificial molecular machines inspired by biology and with diverse functions and applications, including information storage,5 targeted drug delivery,6 mechanical work7 and even the ability to switch “on” and “off” catalytic activity.8 Such naturally inspired molecular machines represent a new and exciting branch of biomolecular engineering—in the words of Leigh himself “that’s how biology does it, so why can’t we?”2.

While the genetic elements contributing to the salinity tolerance of Listeria monocytogenes have been well characterized, the regulatory signals and responses (genetic and/or biochemical) that govern these mechanisms have yet to be elucidated. Encoded by betL , the first genetic element to be linked to listerial osmotolerance, the secondary betaine uptake system BetL is a member of the betaine-carnitine-choline transporter family. Preceded by consensusσ A - and σ B -dependent promoter sites, betL is constitutively expressed and transcriptionally up-regulated in response to salt stress. The nisin-controlled expression system was used to achieve salinity-independent, controlled betL expression in Listeria . In the absence of NaCl-activated transcriptional control, BetL activity was found to be a function of environmental salinity, showing optimal activity in buffer supplemented with 1 to 2% NaCl (osmolality, 417 to 719 mosmol/kg). In addition, BetL was activated rapidly (half-life, 2 min) in response to an osmotic upshift imposed by adding 2% NaCl to 50 mM potassium phosphate buffer.

Listeria monocytogenes is a gram-positive food-borne pathogen that is highly resistant to osmotic stress (NaCl concentrations of up to 10%) and can grow at refrigeration temperatures (31). This characteristic growth and survival under such adverse environmental conditions is attributed mainly to the accumulation of the organic compounds glycine betaine (N,N,N-trimethylglycine) (16, 18) and carnitine (β-hydroxy-γ-N-trimethylaminobutyrate) (3). Accumulated to high internal concentrations without adversely affecting vital cellular processes, these compounds are often referred to as compatible solutes (32). In general, compatible solutes are small, highly soluble molecules which carry no net charge at physiological pH and function to stabilize protein structure and function while also maintaining cell volume at elevated osmolarity (14, 27). As well as glycine betaine and carnitine, other compounds, such as sugars, amino acids, amino acid derivatives, sulfate esters, and small peptides, have been shown to function as effective compatible solutes in bacterial cells (14). For L. monocytogenes cells grown in complex media with 7.5% added NaCl, increases in glycine betaine and carnitine levels were accompanied by elevated concentrations of potassium, glutamate, glycine, alanine, and proline (18). However, while proline at extracellular concentrations of

Despite remaining apparently frozen through the millennia, the genetic code is far more flexible than previously believed and can be extended and repurposed with relative ease.

Survival of the food-borne pathogen Listeria monocytogenes in hypersaline environments is attributed mainly to the accumulation of organic compounds termed osmolytes (57). Osmolytes, often referred to as compatible solutes (9) owing to their compatibility with cellular metabolism at high internal concentrations, can be either transported into the cell or synthesized de novo and act by counterbalancing the external osmotic strength, thus preventing water loss and plasmolysis (14, 15). Beumer et al. (8) identified three principal compatible solutes in Listeria: proline, betaine, and carnitine. While much information is available regarding uptake of these osmolytes from the external environment (8, 40, 53), detailed analysis of osmolyte synthesis systems in Listeria has remained largely unexplored. Unlike the recently identified transport systems BetL (47, 48), OpuC (19; R. D. Sleator, unpublished) and GbuABC (21, 30), osmolyte synthesis is not restricted by the availability of external osmolytes, a factor which may represent an important limitation for growth in hostile environments of elevated osmolarity such as the macrophage phagosome. Optimal growth of L. monocytogenes in low aw environments may thus depend on osmolyte synthesis in combination with uptake. Perhaps the best-characterised bacterial osmolyte synthesis system is that of proline (5, 24, 32). For the majority of bacteria, proline is synthesized from glutamate by a four-step reaction catalyzed by gamma-glutamyl kinase (GK; proB product; EC 2.7.2.11), γ-glutamyl phosphate reductase (GPR; proA product; EC 1.2.1.41), and Δ1-pyrroline-5-carboxylate (P5C) reductase (proC product; EC 1.5.1.2). The remaining step, third in the sequence, occurs spontaneously (12). In other genera, the proB and proA genes generally constitute an operon, which is distant from the proC gene on the chromosome. In addition to this pathway, a number of bacteria have been shown to synthesize proline via offshoots of the arginine biosynthetic pathway (5). As well as its role as an osmoprotectant, recent evidence suggests that proline may function as a virulence factor for certain pathogenic bacteria (6, 16, 46). Marquis et al. (35), using an uncharacterized listerial proline auxotroph obtained following transposon mutagenesis, concluded that while proline auxotrophy had no effect on virulence following intravenous inoculation, the possibility of reduced virulence during the intestinal phase of natural infection could not be ruled out. In this communication we describe the isolation, characterization, and disruption of the listerial proBA operon and investigate the role of this genetic element in contributing to the growth and survival of L. monocytogenes in environments of elevated osmolarity and during subsequent infection (both intraperitoneal and peroral) of a murine model.

Perhaps best known as the genomics pioneer who defied all the odds to decode the book of life and publish its contents years ahead of the government funded Human Genome Project’s projected completion date; J. Craig Venter has done it again – this time in the nascent field of synthetic biology.

Albert Einstein once said that "The true value of a human being can be found in the degree to which he has attained liberation from the self". For years our traditional view of 'self' was restricted to our own bodies; composed of eukaryote cells encoded by our genome. However, in the era of omics technologies and systems biology, this view now extends beyond the traditional limitations of our own core being to include our resident microbial communities. These prokaryote cells outnumber our own cells by a factor of ten and contain at least ten times more DNA than our own genome. In exchange for food and shelter, this symbiont provides us, the host, with metabolic functions far beyond the scope of our own physiological capabilities. In this respect the human body can be considered a superorganism; a communal group of human and microbial cells all working for the benefit of the collective - a view which most certainly attains liberation from self.

Functional environmental screening of metagenomic libraries is a powerful means to identify and assign function to novel genes and their encoded proteins without any prior sequence knowledge. In the current study we describe the identification and subsequent analysis of a salt-tolerant clone from a human gut metagenomic library. Following transposon mutagenesis we identified an unknown gene (stlA, for “salt tolerance locus A”) with no current known homologues in the databases. Subsequent cloning and expression in Escherichia coli MKH13 revealed that stlA confers a salt tolerance phenotype in its surrogate host. Furthermore, a detailed in silico analysis was also conducted to gain additional information on the properties of the encoded StlA protein. The stlA gene is rare when searched against human metagenome datasets such as MetaHit and the Human Microbiome Project and represents a novel and unique salt tolerance determinant which appears to be found exclusively in the human gut environment.

Survival of the food-borne pathogen Listeria monocytogenes in environments of elevated osmolarity and reduced temperature is attributed, at least in part, to the accumulation of the trimethylammonium compound glycine betaine. Previously we identified betL , a gene encoding the secondary glycine betaine transporter BetL, which we linked to the salt tolerance of Listeria . In this report, we demonstrate that betL , preceded by a consensus σ B -dependent promoter, is regulated by osmotic up-shock, at least in part at the level of transcription. Using allelic exchange mutagenesis we constructed an in-frame deletion in betL , and used this mutant to determine the role of BetL in contributing to the growth and survival of L. monocytogenes , both in a high risk food (Camembert cheese) and animal model. Our results indicate that while BetL plays an important role in glycine betaine mediated osmoprotection, mutating the gene does not significantly effect either the cryotolerance or virulence of the organism.

Tuberculosis globally results in almost 2 million human deaths annually, with 1 in 4 deaths from tuberculosis being human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS)-related. Primarily a pathogen of the respiratory system, aerobic Mycobacterium tuberculosis complex (MTBC) infects the lungs via the inhalation of infected aerosol droplets generated by people with pulmonary disease through coughing. This review focuses on M. tuberculosis transmission, epidemiology, detection methods and technologies.

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem.

In only a few short decades molecular biology and genetic engineering have spawned today's burgeoning biotechnology industry—arguably one of the fastest growing and most commercially lucrative bran...

The rationale for choosing pathogens over commensal species as a source of potential stress survival mechanisms is based on a number of factors; not least of which is the fact that certain pathogenic bacteria, with lifecycles bridging the host and external milieu, have evolved sophisticated stress management strategies enabling them to succeed in more diverse ecological niches than those normally occupied by most non-pathogenic species [7]. Pathogens thus represent a rich source of genes which could potentially improve the physiological vigor of less versatile probiotic strains, both external to and within the host.

Background We investigate the role of the C-terminal coiled coil of the secondary proline porter ProP in contributing to Cronobacter sakazakii osmotolerance. Findings The extended C-terminal domain of ProP1 (encoded by ESA_02131) was spliced onto the truncated C-terminal end of ProP2 (encoded by ESA_01706); creating a chimeric protein (ProPc) which exhibits increased osmotolerance relative to the wild type. Conclusions It appears that the C-terminal coiled coil domain tunes ProP at low osmolality, whereas ProP transporters lacking the coiled coil domain are more active at a higher osmolality range.

In the News story “The bacteria fight back” (Special Section on Drug Resistance, 18 July, p. [356][1]), G. Taubes describes the ongoing war between bacteria and antibiotics, which the bacteria appear to be winning. Against this backdrop, scientists are struggling to uncover viable therapeutic

The trimethylammonium compound glycine betaine ( N , N , N -trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon Listeria monocytogenes . We report the identification of betL , a gene encoding a glycine betaine uptake system in L. monocytogenes , isolated by functional complementation of the betaine uptake mutant Escherichia coli MKH13. The betL gene is preceded by a consensus ς B -dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions. BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group. The observed K m value of 7.9 μM for glycine betaine uptake after heterologous expression of betL in E. coli MKH13 is consistent with values obtained for L. monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes . This mutant is also unable to withstand concentrations of salt as high as can the BetL + parent, signifying the role of the transporter in Listeria osmotolerance.

“Your mission should you choose to accept, is to locate the enemy, silently dock on the cell surface and inject the genetic blueprint. Once inside, you must then hijack the host’s cellular machinery and assemble multiple clones. Working quickly then, slice open the cell wall perimeter and escape to repeat the process on the remaining enemy hosts” Although farfetched, this passage describes the efficient killing machinery of bacteriophages, and depicts the events which occur millions of times in nature every day as bacteria succumb to their tiny deadly nemeses.

Recently we reported a role for compatible solute uptake in mediating bile tolerance and increased gastrointestinal persistence in the foodborne pathogen Listeria monocytogenes1. Herein, we review the evolution in our understanding of how these low molecular weight molecules contribute to growth and survival of the pathogen both inside and outside the body, and how this stress survival mechanism may ultimately be used to target and kill the pathogen.

Gastrointestinal disease is a major cause of morbidity and mortality worldwide each year. Treatment of chronic inflammatory gastrointestinal conditions such as ulcerative colitis and Crohn's disease is difficult due to the ambiguity surrounding their precise aetiology. Infectious gastrointestinal diseases, such as various types of diarrheal disease are also becoming increasingly difficult to treat due to the increasing dissemination of antibiotic resistance among microorganisms and the emergence of the so-called 'superbugs'. Taking into consideration these problems, the need for novel therapeutics is essential. Although described for over a century probiotics have only been extensively researched in recent years. Their use in the treatment and prevention of disease, particularly gastrointestinal disease, has yielded many successful results, some of which we outline in this review. Although promising, many probiotics are hindered by inherent physiological and technological weaknesses and often the most clinically promising strains are unusable. Consequently we discuss various strategies whereby probiotics may be engineered to create designer probiotics. Such innovative approaches include; a receptor mimicry strategy to create probiotics that target specific pathogens and toxins, a patho-biotechnology approach using pathogen-derived genes to create more robust probiotic stains with increased host and processing-associated stress tolerance profiles and meta-biotechnology, whereby, functional metagenomics may be used to identify novel genes from diverse and vastly unexplored environments, such as the human gut, for use in biotechnology and medicine.

Bacteria respond to elevated osmolality by the accumulation of a range of low molecular weight molecules, known as compatible solutes (owing to their compatibility with the cells' normal physiology at high internal concentrations). The neonatal pathogen Cronobacter sakazakii is uniquely osmotolerant, surviving in powdered infant formula (PIF) which typically has a water activity (aw) of 0.2 – inhospitable to most micro-organisms. Mortality rates of up to 80% in infected infants have been recorded making C. sakazakii a serious cause for concern. In silico analysis of the C. sakazakii BAA-894 genome revealed seven copies of the osmolyte uptake system ProP. Herein, we test the physiological role of each of these homologues following heterologous expression against an osmosensitive Escherichia coli host.

Against a backdrop of increasing antibiotic resistance, and the emergence of new and evolving pathogens, clinicians are increasingly forced to consider alternative therapies - probiotics are one such alternative.