Microbes and viruses

In 1884, the Danish doctor Christian Gram tried to enhance contrast for light microscopy of bacteria by staining them. He created a staining procedure, still in use today, which split bacteria into stained and unstained bacteria, the Gram-positives and the Gram-negatives.

Coloration is not a faithful way to tell microbes from one another. However this method was reliable enough for almost a century to differentiate the major groups of bacteria. We now know that the ultrastructural properties (shape seen using microscopes and electron microscopes), as well as metabolic and recently genome sequences are much more reliable to tell about relationships between cell types. The Gram-positive organisms' envelopes are made of a single membrane, sometimes very fragile (it is just the lipid bilayer surrounding the cytoplasm of the cell), but usually protected by a strong layer of more or less plane structures made of carbohydrates and short peptides, the murein (or peptidoglycan). In bacteria such as B. subtilis, this envelope comprises as many as forty layers (remember how strong paper is difficult to tear apart, when it is multilayered).

For those interested in scientific debates, there is a rampant but interesting controversy about the origin of bacteria. When bacteria were first identified, more than one century ago, it rapidly appeared that they looked different from the plant or animal cells. They were lacking a well defined nucleus. For this reason the living kingdoms were split into two families, the prokaryotes (from προ, anterior and καρυον, kernel) and eukaryotes (from ευ: well, good, and καρυον, kernel). In 1977, Carl Woese, at the University of Illinois at Urbana, proposed that in fact bacteria were split into two domains, that were as remote from each other as from animal or plant cells, and he proposed to name these bacteria eubacteria (now Bacteria), and archaebacteria (now Archaea). The latter were supposed to have archaic features reflected in their general structure and habitats (usually extremely hot water in volcanoes or in deep sea rifts). While most data (in particular the structure of their ribosomes, the organelles that make proteins) substantiate this difference, analysis of metabolism often does not. This led several scientists, and in particular Dr. R. S. Gupta in Canada to revert to the prokaryote/eukaryote dichotomy, and to split the bacteria into monoderms (with a single envelope) and diderms (with two membranes), roughly covering the Gram coloration dichotomy. Curiously enough, the debate surrounding this discussion is often quite bitter. There seems to be no doubt that the core of the translation transcription machinery is different in Archaea and in Bacteria, but it is also clear that lateral gene transfer is a ubiquitous phenomenon, that will affect relationships between organisms. It would be interesting to understand why there is so much emotion in a debate which, after all, is not without interest: is there only one common ancestor to all forms of life? If yes, what is it, if no, how were constructed the extant organisms?

For nomenclature see List of Prokaryotic Names with Standing in Nomenclature (LBSN)

• Gram-negatives

				These bacteria form straight rods from 0.5-1 to 1.5 micrometer in diameter and from 2 to 6 nanometer in length, depending on the growth phase and environment. Mutants can make spherical cocci, or long filaments. They are usually motile, with peritrichous flagella. The can grow both in the presence and in the absence of oxygen, and from 15-18°C to 46°C. Optimum growth temperature is 37°C, and they are normal commensals of man. They grow well on bile salts, which is why they colonize easily the intestine (and sometimes the gall bladder and even the liver). This is used as a criterium to differentiate all Enterobacteria (bacteria living in the intestine of animals) from other bacteria. Their shortest generation time can be 18-20 minutes, which make them among the fastest growing bacteria. Most E. coli strains can grow on the milk sugar lactose. Several thousand laboratories work on Escherichia coli, which is the best known living organism. Thousands of research articles have been published on this organism or using it. It is now commonly used as a genetically modified organism to produce foreign proteins (even human ones, such as the Human Growth Hormone) or metabolites. The major rules of gene expression have been discovered by scientists working with this organism, or with its viruses, known as bacteriophages (or phages, for short). In the '30s bacteria were thought to differ from animal and plants, in that they mutated very fast, for this reason E. coli was usually named Escherichia coli mutabile. Bacteria were then thought to be refractory to genetic studies. It was only when Oswald Avery discovered that bacterial DNA was the carrier of genetic heredity that bacterial genetics really began. One of the first strain, ML (for Monsieur Lwoff, but some say "merde de Lwoff", meaning that it was isolated from André Lwoff stools...), was studied at the Institut Pasteur. And it is there that Jacques Monod and François Jacob unraveled the question of bacterial adaptation to their environment. They demonstrated that many genes were only expressed after an appropriate environmental trigger started their synthesis, through a series of steps involving RNA synthesis (transcription) and protein synthesis (translation). The genome sequence of two reference strains has been deciphered, that of strain MG1655, by an American team, and that of W3110 by a Japanese team. Many E. coli strains are pathogenic and toxinogenic. In particular one can recognize an enterohaemorrhagic strain causing very dangerous outbreaks (O157:H7: without pili "Ohne Haar") or RS218, causing meningitis The EcoR reference collection provides strains from all over the world which can be used to analyse the evolution of E. coli.
	Escherichia coli
		Reference K12 strain collection: CGSC Reference gene annotation: EcoGene Reference genome database: Colibri

• Helicobacter pylori

In 1982, the Australians Barry Marshall and Robin Warren discovered the presence of bacteria in the stomach of patients affected by chronic gastritis. First named Campylobacter pylori (because of their superficial similiarity with a frequent pathogen of the intestine, Campylobacter jejuni) these bacteria shaped as cork screws were renamed Helicobacter pylori. From the study of the pathological phenomena induced by H. pylori in the stomach, Marshall et Warren proposed the bold hypothesis at that time, that these bacteria are not simple opportunistic commensals of the stomach of affected persons, but are actually the cause of chronic gastritis and ulcer. They reached this conclusion because it was possible to demonstrate the presence of these bacteria in all patients affected by duodenum ulcers, and in 80% of the patients with gastric ulcer. Furthermore, the 20% remaining patients with gastric ulcer had used large quantities of anti inflammatory drugs such as aspirin or ibuprofen, which are known to provoke ulcers of the gastric membrane, and this accounted well for the disease in their case.
But, in spite of the clarity of the conclusions of Marshall et Warren (in particular when after having tried on themselves infection by H. pylori they witnessed the apparition of gastritis) the demonstration that H. pylori is the cause of ulcer took more than ten years to be accepted, in particular because of the implicit resistance of pharmaceutical companies that produced anti-ulcer drugs. Indeed, gastritis and ulcer are "ideal" diseases, in that they rarely kill, or very slowly, while they create a need for a chronic treatment. In addition, because the studies with humans (in particular double blind studies, involving control groups treated with a placebo, and groups treated with experimental drugs) are extremely costly, they are often supported by grants from industry. It is therefore very easy to prevent or slow down research simply by refusing to finance those types of experiments that would go aginst the interests of the companies. We had therefore to wait for a study financed by the NIH to demonstrate the causal relationship between H. pylori and ulcer, in 1994. This study showed the ubiquitous presence of the bacteria in almost all patients, and that one demonstrated that the action of appropriate antibiotics lead to curing the disease. I was, finally accepted that the bacteria are its real cause.
What is kown at present about this infection? Helicobacter pylori are bacteria often found in the stomach of mammals. It has been shown experimentally in animals that it provokes a chronic inflammation and ulcers (in parallel with the acidic secretion of the stomach). H. pylori strives in this environment extremely hostile by its acidity, because it produces an enzyme, urease. Urease degrades urea produced by the bacterial metabolism and produces ammonia that neutralizes the stomach acid. This which allows bacteria to survive. Perhaps because of their helicoidal shape (hence their name) and of the way they move, these bacteria are able to penetrate the mucous cover that protects the stomach epiderm. Furthermore they degrade the mucus cover of the stomach, and reach the cells of the gastric wall that become sensitive to the proteolytic enzymes that are normally secreted in the stomach (helping digestion), such as pepsin. The bacteria can also adsorb to the surface of the gastric cells, provoking inflammation. Finally, for reasons that are not yet fully understood — and that one hopes to understand with the analysis of the genome sequence — H. pylori also enhances the acid secretion of the stomach. This triggers inflammation of the first duodenum. In some persons this leads to production of abnormal cells (displasy). Helicobacter pylori then attacks these cells, and this leads to ulcer, and sometimes to cancer.
Thus, after a few weeks of infection by H. pylori, most people develop gastritis. Fortunately, most people never get morbid symptoms, and do not suffer any trouble due to the infection. Nobody knows yet why some person react, and others do not. It is most likely that genetic factors (in particular associated to the nature of the surface of cells) and of the environement (such as situations of stress), favour H. pylori development, and the associated diseases. As an outcome of genome programs, it appears that there exists various more or less pathogenic H. pylori strains. Finally, one does not understand very well the route for contamination. There exists a strong correlation between the social status, the ethnic group, and age (the frequency of contamination increases with age and lack of hygiene). However contamination often happens during infancy, and bacteria inhabit their host all his or her life. Understanding the meaning of its genome text might help develop a vaccine (by analyzing the toxins or the molecules present at the surface of the bacteria), or to find targets for new sepcific antibiotics acting in acidic conditions.

Helicobacter foundation

Proteomics of Helicobacter

The Helicobacter pylori genome database, PyloriGene

• Photorhabdus luminescens

As E. coli, a member of Enterobacteriaceae, see programme of the HKU-Pasteur Research Centre, which colonizes the gut of nematode worms, and kills insects.

The lifestyle of this organism splits into two host-associated stages, with no known free-living stage. In the first symbiotic stage bacteria colonize the intestinal tract of soil nematodes of the Heterorhabditis genus. In a second, pathogenic, stage the couple bacteria-nematode invades and kills a wide variety of insect larvae. The nematodes regurgitate their bacterial symbionts into the insect circulatory system (haemocoel), where P. luminescens proliferates rapidly, overcoming the insect immune response. The antimicrobial response of insects comprises both cellular and humoral reactions. Plasmatocytes, the major haemolymph cells, exhibit strong phagocytic activity. They also trigger a first line of defense composed of highly toxic molecules: nitric oxide (NO), superoxide and hydrogen peroxide (H₂O₂) and peroxynitrite (ONOO^–), rapidly generated from the reaction between NO and superoxide. Associated to the generation of reactive oxygen species, melanization also contributes to the insect host defence. The humoral reaction results from induction by microbial challenge of antimicrobial peptide genes in the fat body, followed by the secretion of these peptides into the haemolymph. In Drosophila seven distinct inducible antimicrobial peptide families have been described. They are structurally diverse and mostly small in size (ca 5 kDa), cationic and membrane targeted. Their activity spectra are directed either against fungi (Drosomycins, Metchnikowin), or against Gram-positive (Defensin) and Gram-negative (Attacins, Cecropins, Drosocin, Diptericins) bacteria. It is assumed that their combined activities blocks the growth of invading microorganisms. The processes by which P. luminescens circumvents all these host defense systems are not yet well characterized: as perhaps could be expected from the virulence of low infection inocula, the protease and lipase fractions that might degrade antibacterial molecules of the insect immune response do not significantly affect mortality rates and a strain deficient in PhlA hemolysin still exhibits a high virulence. In contrast, the way by which P. luminescens kills the insect is better known. As they multiply, the bacteria produce many toxins, such as the Mcf (Makes Caterpillars Floppy) toxin, a dominant virulence factor critical to pathogenesis and the four toxin complex forms, Tca, Tcb, Tcc and Tcd exhibiting a high toxicity to Manduca sexta. In parallel, they also produce various antibiotics inhibiting the growth of competing microorganisms in the insect cadaver and enhance conditions for nematode reproduction by providing nutrients and growth factors. Finally, after several rounds of reproduction, a new generation of infective juvenile nematodes reacquire the bacteria and then leave the insect carcass.

The database PhotoList summarizes all information available on the genome of P. luminescens ssp laumondi TT01.

• Salmonella typhi

This bacterium is very similar to its cousin E. coli. It has the same overall shape and general metabolic properties. In contrast to E. coli it cannot normally grow on lactose.

Typhoid fever is a dangerous gut infection. The first symptoms are similar to those of a bad cold, or of flu, with headache, sore throat and fever, slowly increasing to 40°C or more. Usually the pulse is lower than would be expected for such a high fever, and it may even get lower as fever increases. These symptoms are associated to nausea and diarrhoea (sometimes they may lead to constipation). After one week some red patches appear on the body. Associated to diarrhoea the patient becomes weaker and weaker, looses weight and get dehydrated. The course of the disease then may become under control or, often, get worse, with blood in the stood, peritonitis and many other complications including pulmonary ones. If the disease is diagnosed early enough it may be cured using appropriate antibiotics. It is nevertheless a highly contagious disease, and the patient should be isolated as much as possible, with the persons taking care instructed to follow drastic hygiene rules.

• Shigella flexneri

Genome studies confirm that Shigella and Escherichia constitute a same species. Shigella bacteria are therefore pathogenic E. coli strains, which usually carry a virulence plasmid, with genes coding for toxins. Shigella are non motile as are many bacterial pathogens. They cause in man and other primates bacillary dysentery. The infection is usually restricted to the large bowel and the rectum, with acute inflammation and ulceration. Usually the infection, although severe, is self-limiting. Shigella sp are usually sensitive to usual antibiotics, but resistant forms are spreading. Several species are defective in some of the polyamines (they do not make cadaverine) and this appears to be related with virulence.

The El Tor N16961 genome sequence at TIGR

• Yersinia pestis

Plague or Black Death was perhaps the disease which took the heaviest toll on humanity. This disease was described during Antiquity, it then reappeared in the Middle Ages, and swept through Europe where it killed perhaps one third of the European population. Accounts of one of the latest outbreaks can be found in the very interesting diary of Samuel Pepys (1633-1703; in which one finds all sorts of comments about life and ideas in England at that time). A third pandemics began in the latter half of the nineteenth Century, and spread though North Africa and Southern Europe, as well a through most of the world, where it still has foci of endemy (Iran, Madagascar, China, and even California). Of course, the outbreak in Hong Kong is still in the memory of people there, and it is Alexandre Yersin who isolated and characterized the bacteria there in 1894. A vivid account of plague in Algiers is given by Albert Camus (1913-1960; La Peste). The final sentence of the book: "Écoutant, en effet, les cris d'allégresse qui montaient de la ville, Rieux se souvenait que cette allégresse était toujours menacée. Car il savait ce que cette foule en joie ignorait, et qu'on peut lire dans les livres, que le bacille de la peste ne meurt ni ne disparaît jamais, qu'il peut rester pendant des dizaines d'années endormi dans les meubles et le linge, qu'il attend patiemment dans les chambres, les caves, les malles ,les mouchoirs et les paperasses. et que, peut-être, le jour viendrait où, pour le malheur et l'enseignement des hommes, la peste réveillerait ses rats et les enverrait mourir dans une cité heureuse. " is a black reminder of a bleak future.

The reservoir of the causing agent is small mammals, often rodents, sometimes bats. And it was a saying that when people began to see dead rats in the streets or nearby villages, this was a sign that the disease would appear. This is because the vectors are different species of fleas, normally living on rodents, which, when the animal die out, need to find another host, man in particular. Usually the vector fleas are repulsed by the smell of horses. This explains why, in the Middle Ages, it was found that people ordinarily dealing with horses never caught the disease: they did not wash themselves at that time, and, being impregnated with horse's smell, they were repulsive to fleas... This is another example of the tight link between social behaviour and the spread of infectious diseases.

The genome sequence of Y. pestis has been established at the Sanger Centre. Remarkably, it is smaller than that of E.coli and that of its parent Y. pseudotuberculosis. Even more remarkable is the fact that many genes are inactivated in the genome, so that they are certainly not active for pathogenicity. In fact, the study of the Y. pestis genome demonstrates that loss of function may yield increased pathogenicity. Analysis of the sequence of a pathogenicity island (i.e. a cluster of genes involved in pathogenicity), by Buchrieser, Glaser, Kunst and Carniel showed that there are differences between the strains thought to be responsible for the major pandemics, Y. pestis pathovar antiqua, mediaevalis, orientalis. In fact this study demonstrates that Y. pestis is still evolving, and that this evolution leads it to lose more and more gene functions, as some genes which were still apparently functional in pathovar antiqua, are no longer so today. In addition to genes present in its chromosome, a large plasmid is necessary to establish virulence of the bacteria.

The genome sequence at the Sanger Centre

• Gram-positives

			The ultimate goal of any living organism is to occupy a part of the Earth crust. This means, among other ancillary functions: exploration, colonization, maintenance and exploitation of the local resources dealing with congeners and with other organisms, etc. As a consequence, one cannot understand an organism if one does not have an idea about its habitat. B. subtilis was first identified in 1872. It is a bacterium that can be routinely obtained in pure culture by soaking hay in water for a few hours at 37°C, then filtering and boiling for one hour at neutral pH. Bacillus subtilis has also been isolated directly from soil-inoculated nutrient agar, where B. subtilis predominates among the outgrowing cultures. Spores are more readily obtained in solid media than in liquid media, and they require the presence of manganese ions. The bacteria produce a complex lipopeptide, surfactin that permits them to glide very efficiently over the surface of certain types of media. This property is likely to be related to colonization of the surfaces of leaves (the phylloplane), fruits or sometimes roots. Indeed B. subtilis makes up the major population of bacteria on flax stems during the retting process. Vegetative cells of B. subtilis are responsible for the early stages of breakdown of plants, and sometimes products of animal origin; some variants cause potato tubers to rot (B. amyloliquefaciens). When conditions become unfavorable, the onset of a differentiation process, sporulation, permits the cells to generate resistant spores that can be easily dispersed throughout the environment where they will germinate if conditions are appropriate. Unlike most other bacterial species, endospore-forming bacteria are highly resistant to the lethal effects of heat, drying, many chemicals and radiation. In fact, one fashionable hypothesis of the origin of life on Earth by panspermia (Sven Arrhenius, and more recently Francis Crick) rests on the notion that bacterial spores such as thoses of B. subtilis could travel through space and survive for millions of years. Despite its nice appeal to wild imagination, this hypothesis essentially puts the investigation of the origin of life out of our reach… The genome of Bacillus subtilis was the first one to be known of this category of organisms. It substantiated the idea that its biotope is indeed the phylloplane. Bacillus subtilis is not an entirely obligatory aerobe, since it can grow in the presence of electron acceptors other than oxygen. But it requires usually a high oxygen pressure to grow optimally (this is in line with its growth on the surface of leaves). This bacterium is larger that E. coli, typically it is a rod 1.5 micrometer wide and 3 micrometers long. more...
Bacillus subtilis
	Go to the reference genome database: SubtiList

• Bacillus anthracis

Bacillus anthracis is a Gram positive organism very similar to the Bacillus cereus complex (which comprises in particular insect pathogens, under the generic name Bacillus thuringiensis). It is also displaying many characters present in Bacillus subtilis. In the same way as these bacteria B. anthracis is particularly resistant under its dormant "seed" form, heat resistant spores. The spore can survive easily 100 years, and spores more than 200 years old have been germinating. This is the reason why some fields have frequently been thought to be cursed: they had seen infected animals, and years after, naive animals caught the disease in those fields. It is non motile (as many pathogens) and possesses a capsule which makes it interact with animal cells efficiently. Sporulation requires a temperature lower than 42°C and the presence of oxygen, and this is the reason why it is generally forbidden to make the autopsy of corpses from animals infected by anthrax: bacteria multiplying inside the host will die without sporulating, making the risk of contamination low. Finally the bacteria contain a minichromosome (virulence plasmid) which carries three important genes: the lethal factor (LF) gene, the edema factor (EF) gene and the protective antigen (PA) gene. The lethal factor is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase (a control enzyme essential for the cell's life) and causes lysis of macrophages. The protective antigen binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol. It is therefore needed to allow the former toxins to penetrate the host cell. The edema factor gene is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis, and it has been cloned and identified using an original method. Bacillus anthracis causes anthrax (charbon, in French) a disease that exists under three forms. Cutaneous anthrax is a skin lesion with a black centered ulcer (hence the name). It can be easily cured. Two forms are extremely dangerous, inhaled anthrax and digestive anthrax. Spores are engulfed by white cells, where they germ and rapidly multiply instead of being destroyed. The toxins create a strong edema and necrosis of the tissues, associated to hemorrhagies. They kill the host even if the bacteria have been killed by antibiotics. The only way to cure the disease is to have a very early diagnostic, a difficult requirement since the disease begins with symptoms similar to those of many respiratory or digestive infectious diseases. Under natural conditions anthrax (usually cutaneous anthrax) is a professional disease associated to the work of persons dealing with animals or leather. Bacillus anthracis has unfortunately been used as a warfare agent in particular by the UK, which exploded a bomb on Gruinard Island (with difficult problems of decontamination years after) and the former Soviet Union (with an identified accident in the region of Sverdlovsk. A live vaccine (Sterne vaccine) exists, that uses a non capsulated strain. Usually one uses an acellular vaccine which is composed of the toxin elements associated to appropriate factors. It is used for personnel exposed to anthrax, following a very heavy protocol (at least six injections). Its efficacy is lower than that of the live vaccine.

The Bacillus anthracis genome database: AnthraList

• Enterococcus
• Mycobacterium leprae

  					The agent of leprosy is a strongly acid-fast rod-shaped organism with parallel sides and rounded ends. In size and shape it closely resembles the tubercle bacillus. It occurs in large numbers in the lesions of lepromatous leprosy, chiefly in masses within the lepra cells, often grouped together like bundles of cigars or arranged in a palisade. Chains are never seen. Most striking are the intracellular and extra-cellular masses, known as globi, which consist of clumps of bacilli in capsular material. Under the electron microscope the bacillus appears to have a great variety of forms. The commonest is a slightly curved filament 3-10 m in length containing irregular arrangements of dense material sometimes in the shape of rods. Short rod-shaped structures can also be seen (identical with the rod-shaped inclusions within the filaments) and also dense spherical forms. Some of the groupsof bacilli can be seen to have a limiting membrane. It is believed that only leprosy bacilli which stain with carbol-fuchsin as solid acid-fast rods are viable and that bacilli which stain irregularly are probably dead and degenerating. The differences are valuable pointers in biopsy specimens to the effects of treatment. In patients receiving standard multidrug therapy (MDT), a very high proportion of bacilli are killed within days, which suggests that many of the manifestations of leprosy, including reactions ofthe erythema nodosum type, which follow initial treatment, must be due in part to antigens from dead organisms rather than living bacilli. We therefore need drugs which will help the body to dispose of dead but still intact leprosy bacilli. The genome sequence of the bacillus, coordinated by Pr Stewart Cole, revealed that it comprises a large number of genes in the process of being inactivated. This accounts for the fastidious nature of the organism, and for the fact that it has never been possible to cultivate it, except on live nine-banded armadillos.
  			(Rights Reserved)
  		Reference genome database: Leproma

• Mycobacterium tuberculosis
• Staphylococcus aureus
• Staphylococcus epidermidis

The SepiList database

• Streptococcus pneumoniae

This organism is famous. It is with S. pneumoniae that Oswald Avery and his colleagues, in 1944 demonstrated that DNA was carrying genetic information. This experiment was made possible because this bacterium is competent for transformation (the phenomenon of change in genotype mediated by incorporation of exogenous DNA into a genome).

• Streptococus pyogenes

This member of the Streptococcus genus group A is named the "flesh eating bacterium". This group of Gram positive bacteria is very common and causes a wide variety of diseases from benign infections such as common angina or tooth infection to the usually more dangerous scarlet fever. But they are the cause of fortunately rarer diseases which are extremely dangerous, such as toxic shock.

The genome sequence has been obtained at Oklahoma University.