Small Things Considered

The Gram Stain: Its Persistence and Its Quirks

by Elio

What is more emblematic of our science than the Gram stain? Since its invention 130 years ago, it has been in frequent and continuous use. It conveniently places most bacteria into one of two groups, the Gram-positives or the Gram-negatives. Gram staining is cheap, effective, quick, and relatively easy to interpret. Its most useful application is in the clinical setting. When examining a smear of, say, pus from an abscess, this stain often allows to include for consideration roughly half the clinically relevant bacterial species while excluding the others. Or consider a patient with meningitis. Here, speed is of the essence because treatment must be initiated right away. A Gram stain of the spinal fluid may reveal within minutes the presence of Gram-positive cocci, (probably pneumococci), Gram-negative cocci (almost certainly meningococci), or Gram-negative slender rods (most likely Haemophilus influenzae). This can make a critical difference in the choice of antibiotics that have to be administered in great haste. However, when it comes to characterizing the bacteria in an environment, its usefulness diminishes, in part because it is not always in step with taxonomy, which I’ll discuss below. And there yet is another side to this story. Quite a few bacterial species that stain positive early in the growth of a culture become Gram-negative later on. Does this detract from the value of Gram’s method? It may, but not in the hands of a person experienced in its use.

A Whiff of Taxonomy – Verrucomicrobia, The Bacterial Warthogs

by Elio

"We occasionally post very brief taxonomic pieces on selected bacterial groups. Be warned that we may not be fully attentive to the taxon level and may mix up genera and higher taxa."

A Verrucomicrobium. Source.

Unlike warthogs, likely to be considered beautiful only by their mother, the Verrucomicrobia  (verruca means “wart, thus the warty bacteria; more about this later) have considerable appeal, be it morphological, physiological, or ecological. This is yet another phylum that owes its recognition to nucleic acid technology. Although few of its members have been cultivated, 16S rRNA studies have confirmed that they are widely distributed in many environments, such as fresh water, oceans, soils, and even vertebrate feces.  Some are host-associated, their hosts including humans and, just because you need to know it, their proportion in the gut of ground squirrels increases as they hibernate. There are no known pathogens in this group although suspicion has arisen.  

Why Listeria Is Competent to Be Virulent

by S. Marvin Friedman

A listeriosis outbreak of 2011 caused 147 cases of illness and 33 deaths in 28 states in the US. The source was cantaloupes grown at one Colorado farm. Source.

It is downright scandalous that in our hi-tech world food-borne infections should be so prevalent (some 48 million cases a year in the US alone, with about 3000 deaths). The tools to take care of these problems are hardly mysterious, requiring mainly safe food production and preservation. High on the list of bacterial offenders is Listeria monocytogenes, a Gram-positive facultative intracellular pathogen that is acquired through ingestion of contaminated food or fluids. Listeria is found in uncooked meats, vegetables, fruits such as cantaloupes, unpasteurized milk and milk products, and some processed foods. Although pasteurization and sufficient cooking will kill Listeria, contamination often occurs after cooking and before packaging. Listeriosis is a disease primarily affecting pregnant women, newborns, adults with compromised immune systems, and the elderly. The two most common life-threatening symptoms are sepsis and meningitis. This is a serious disease with a relatively high mortality that can reach 25%.

L. monocytogenes is an intracellular parasite of epithelial cells and macrophages. It initially resides in a membrane-bound vacuole, the phagosomal vesicle, which is a dangerous site for many bacteria. In order to carry out a successful infection, it escapes into the host cell’s cytoplasm by lysing the vacuolar membrane via a pore-forming hemolysin, listeriolysin and two phospholipases. However, the precise mechanism of this escape is not fully understood. Once within the host cell cytoplasm, the bacteria replicate and use the host’s actin filament network to propel themselves within the cell and from cell to cell.

The Excitement of Clinical Microbiology

by Elio

Members of the San Diego VA Clinical Microbiology lab: (L to R) Romelia Quinonez, Raymond Samson, Carlo Basallo, Monica Beach, Icela Gonzalez, Dr. Joshua Fierer, Juan Ybarra, Jasmine Estrada, Tracey Grosser, Laura Gomez.

Clinical microbiology, one of the major branches of microbiology, goes largely unnoticed by academics, in part perhaps because the diagnostic activities of microbiologists are pursued separately, in hospital and commercial labs. I’d venture to guess that many academic microbiology researchers have never set foot in one of these labs. Their loss, as it would be an exciting and rewarding experience.

This rift casts aside the historical roots of our field. At its beginning, soon after bacteria were found to be the cause of infection, there was a huge rush to develop methods for determining the identity of agents responsible for a patient’s illness. Ingenious laboratory media were designed to favor the growth of certain organisms and to reveal their distinguishing properties, and serological techniques were developed alongside. In those days, there was no significant distinction between diagnostic microbiology and the rest. In time, as research in other fields of microbiology matured, these diverged more and more from this point of origin.

A Kick in the Midgut

by S. Marvin Friedman

Malaria is one of the most devastating infectious diseases in the world today. About 400 million people are infected each year and of those 1.2 million die. Efforts to control malaria have been held back by the lack of an effective vaccine, the alarming rapidity with which Plasmodium, the protozoan parasite, develops drug resistance, plus the failure to eradicate the Anopheles mosquito vector. Fresh approaches to fight malaria are urgently needed, to be used singly or perhaps in combination. One novel approach to a vaccine was recently discussed in this blog.

Formation of Plasmodium berghei (a rodent parasite) oocysts in culture. A. Ookinetes. B. Transforming ookinete and C. young oocysts. F. Transformation begins with a small hump on the outer edge of the ookinete. Transforming ookinetes (“tooks”) then take on a snail-like appearance (v). The entire population of ookinetes transform in 12–36 h, depending on nutrient availability. Source.

Plasmodium undergoes an unusually large number of lifestyle changes in its trip from the female mosquito to a human and back. This is one of the most complex life cycles extant, with so many details that it taxes one’s memory. Each one of the dozen or so stages is labeled with a fancy name and ought be a target for intervention but, for a host of reasons, that has proven elusive. However, the parasite is especially vulnerable in one stage, the transition from a cell called the ookinete to one named the oocyst, which occurs in the mosquito’s midgut. Eventually, the oocysts cross the midgut epithelium into the insect’s circulatory space, the hemocoel. But before going across, the ookinetes are detected by recognition components of the mosquito’s immune system. Killing factors from both the midgut and nearby tissues are recruited and the ookinetes targeted for destruction. In some model systems, fewer that 5% of the ookinetes survive. This, then, is a bottleneck in the Plasmodium life cycle and an attractive place to try to intervene for extermination of the parasite.

Ovobacter propellens, Not Your Average Boring Bacterium

TEM section through an O. pro-
pellens showing the abundant
flagella and a row of regularly
spaced, electron dense organelles
consisting of stacked membranes.
Bar = 1 μm. Source.

by Elio

I bet most of you have never heard of this one. And you are not alone in this. Look for it by name in PubMed and you find two entries, both from the same lab. This bug deserves better. Found by using a fairly direct enrichment technique from sediments in shallow Danish waters, this organism evinces aspects of both structural and physiological uniqueness. It is seen as an ovoid cell that looks less like a bacterium than like a small ciliate. The evidence that it is a prokaryote relies on morphology alone, as it has not been cultivated nor has its DNA been sequenced.

Source.

Ovobacter propellens (or, to reluctantly obey one of my least favorite rules of taxonomy, Candidatus O. propellens) appears as large ovoid cells, 4-5 μm in length, each possessing a huge tuft made up of some 400 flagella. Armed with such equipment, it travels at astounding speeds, having been clocked at 1 mm per second. (That’s some 200 body lengths. To equal that, a human would have to swim at over 300 meters per second.) Now for a small digression: by chance I encountered a most informative table of examples of microbial motility. You will notice (see table) that while paramecia outswim all others in terms of velocity, protists and bacteria alike, O. propellens matches them.

The Three Faces of Thiomargarita

by Merry

How inappropriate to call this planet Earth when it is quite clearly Ocean.
            Arthur C. Clarke

Budding is evident on these attached, elongated Thio-
margarita-like bacteria collected near a methane seep off
the coast of Costa Rica. Bar = ~1 mm. Source.

Ahhh, the delicious aroma of hydrogen sulfide. When as kids we encountered  it, we would hold our nose and proclaim “yuck, rotten eggs!” It is indeed produced by something ‘rotting,’ but specifically rotting under anaerobic conditions, such as in swamps and sewers. It is also abundant in coastal marine sediments, produced by the oxygen-consuming heterotrophic decomposition of organic matter. This makes for a layer at the sea bottom that is sulfide rich but oxygen poor. Nevertheless, leaving no potential energy source untapped, some bacteria in these zones use sulfur oxidation as their energy source. They include two remarkably large γ-proteobacteria, Thioploca and Thiomargarita. Thioploca copes with the lack of oxygen by using nitrate as an alternate terminal electron acceptor, but this just exchanges one problem for another. The sulfides are in the sediment, the nitrate in the water column above. So Thioploca commutes between the two zones, as Elio described in an earlier post.

Thiomargarita namibiensis, the “Namibian sulfur pearl.”
Courtesy of the Microbiological Garden. Source.

Non-motile Thiomargarita uses a different tactic. This bacterium was first discovered in 1999 off the Namibian coast, thus was named T. namibiensis. Its cells are large spheres, arranged in chains, each chain enclosed in a mucous sheath. Average cell diameter is 180 μm, with rare individuals reaching 750 μm, which is about as big as bacterial cells get. But they do this by cheating. Most of the ‘cell’ is a large vacuole, with the cytoplasm relegated to a thin surrounding outer layer. Sulfur globules are evident in the cytoplasm. Thus the genus name, Thiomargarita, that was derived from the Greek for ‘sulfur pearl.’ Thiomargarita spp. are widespread in hydrogen sulfide-rich coastal sediments. They, too, use nitrate as their terminal electron acceptor. Their strategy is to take advantage of the occasional events that resuspend the sediment and bring abundant nitrates into the water column. They then hoard nitrate in their huge vacuoles—enough nitrate to see them through even months of scarcity.

The Paenibacillus Moving Company

by Elio

Enlightenment, a bacterial colony. Displayed in the
Esher Ben-Jacob online art gallery. Source.

Microbes get around. They can be carried by the wind, by insects, or by water currents, sometimes across large distances, sometimes from one grain of soil to the next. Some bacteria contribute to their own dispersal by their motility or, rarely, one may think, by the motility of other microbes. It’s hard to generalize here because there are just too many variables, too many conditions where microbes are on the move. But this business can be pure fun. So it’s with pleasure that I read about a delightful account of how bacteria can transport fungal spores to the benefit of both.

But first a little background, both microbial and personal. Some bacteria, when placed on an agar surface, do not stay put but spread out from the point of inoculation. Certain ones, such as Bacillus mycoides, do it simply by extending their long filaments, but others do it by moving on the agar surface. There are several ways to move along, either with flagella (a process called swarming) or without them (e.g., gliding or twitching). Swarming, whether in Gram-positives like certain Bacillus species or in Gram-negatives such as Proteus, is a communal activity. Single cells don't swarm, aggregates of several cells do. What you see under the microscope are rafts of bacteria arranged side by side, moving along a path that is sometimes regular, sometimes not. To download a movie of a ‘raft’ of ceils swarming together, click here (or to go to the web page where you can select additional file 4, click here). The speed of movement across the agar surface is pretty amazing, about 180 µm/min or 10 mm/h. This means that if you place such a bacterium in the center of a 14 cm Petri dish, it will reach the edge in about seven hours. When such a swarm has covered a certain distance, the cells either stop moving, as in the case of Proteus, or make round disc-like colonies that have the odd ability to rotate. For a movie of a ‘colony’ of cells swarming together, click here (or to go to the web page where you can select additional file 2, click here). The speed at which this rotation happens is also quite fast, one revolution in about 10 seconds. Towards the end of my career, but while I still had a lab, my collaborator Dana Boyd and I played around with these organisms. This was the last time that I worked at the bench, so I quite cherish this topic.

Geobacter: Microbial Superhero

by Suzanne Winter

Iron-breathing microbes, Geobacter. © Derek Lovley
Kelly Nevin & Ben Barnhart, University of Massa-
chusetts. Source.

If the power of Superman resided solely in his supernatural abilities, then Bruce Wayne’s Batman would never have been able to compete on his level. But when the other factors that superheroes depend upon are accounted for—sidekicks, spandex, sarcasm—both Superman and Batman can claim success in the world of do-goodery and hunky haircuts. In other words, the strength and effectiveness of an individual’s power often depends entirely on the creative use of raw materials.

This reality is mirrored in the microbial realm: organisms compete to occupy and dominate specific niches through adaptation and natural selection, and it’s the ones that play by an entirely different set of rules that may have the possibility to change the world.

Notably, species that have evolved innovative and, subjectively, weird methods of living have long intrigued scientists with their survival abilities and preferences. Consider the fascination with the extremophiles in the hot springs at Yellowstone National Park that led to the isolation of Taq DNA polymerase from Thermus aquaticus, surely one of the most important enzymes in the molecular biology lab. And while many microbes follow the well-studied route of using oxygen as a final electron receptor, researchers are increasingly looking at Geobacter species that use iron oxides as the final electron receptor in order to uncover the secrets enigmas of environmental restoration, energy harvesting, and the creation of the world.

Some Like it Hot

by S. Marvin Friedman

Red coloration on rocks near Naples, Italy, produced by the
hyperthermophile Sulfolobus solfataricus. Source.

How can thermophilic bacteria not only survive, but actually proliferate, at elevated temperatures that would be lethal to all other forms of life? After extensive research during the past five decades, this question has been answered in a general way, but the molecular basis for this unusual capability has not been clearly resolved. Thermophiles [I use this term to include both thermophiles (optimal growth temperatures of 50-70 °C) and hyperthermophiles (optimal growth temperatures >80 °C)] synthesize intrinsically thermostable cellular components and/or extrinsic stabilizing factors (chaperonins and polyamines, for example).

Most proteins isolated from thermophiles are thermostable and the mechanisms underlying this property have been extensively studied. These investigations have their roots in the pioneering work on the heat stability of hemoglobin and ferredoxin carried out by Perutz in 1975. Some of the strategies reported to bring about protein thermostability include higher levels of charged amino acids on the protein surface that promote ionic interactions, amino acid preferences, hydrophobic cores, aliphatic side chains, disulfide bridges, and solute accumulation. It is obvious from these studies that a universal mechanism to achieve protein thermostability does not exist. The complexity of this problem is highlighted by differences that appear to be in play for multimeric proteins versus single polypeptide chains, for soluble proteins versus membrane proteins, and for proteins from thermophiles versus proteins from hyperthermophiles.