Small Things Considered

Mysteries of the Bacterial L-Form: Can Some of Them Be Unveiled?

by Hans H. Martin

Figure 1. Peptidoglycan sacculi isolated from L-form
spheroplasts of P. mirabilis. Electron micrograph of
Pt-Ir-shadowed sample. 20,000X.

L-forms are bacterial variants with defective cell walls and irregular growth and multiplication. They arise after peptidoglycan, the exoskeleton of the bacterial cell wall, has been either degraded by bacteriolytic enzymes, or its biosynthesis has been disturbed by antibiotics and other inhibitors, or by defect mutations in essential genes for cell wall synthesis. L-forms with different degrees of wall defects can arise. International experts, headed by nobelist Sidney Brenner, recognized the need to distinguish between entirely cell wall-less protoplasts, surrounded only by a cytoplasmic membrane, and spheroplasts with residual, fragile cell walls. L-forms were discovered in 1935 by Emmy Klieneberger and subsequently described by many authors (examples here and here). Much interest in L-forms arose from their assumed but still unconfirmed roles as concealed pathogens and as survivors of antibiotic action. They are also useful tools for the study of basic mechanisms of cell biology, such as cell division. Yet, as justly deplored in a recent review, L-forms are still "unfamiliar to many microbiologists" and are often regarded "with scepticism." One hears complaints about the unusually labor-intensive and time-consuming process of L-form isolation and cultivation, and the uncertain outcome. However, in my experience, this can be overcome by patient determination.

A special type of resistance to antibiotics, which we studied extensively in my lab, is reflected in the ability of the Gram-negative bacterium Proteus mirabilis to evade the inhibitory action of penicillin and other β-lactam antibiotics by growing as spheroplast- L-forms (references here and here). It is important to note that the most common resistance mechanism, inactivation of β-lactam antibiotics by β-lactamase, is not involved in this phenomenon.

When Crenarchaeota Divide, They Multiply

by Jenn Tsau and Suzy Szumowski*

Sulfolobus acidocaldarius, an extreme thermophile occurs in
geothermally-heated acid springs, mud pots and surface soils;
it can withstand temperatures from 60 to 95 ºC, and a pH of
1 to 5. Left: Electron micrograph of a thin section (X85,000);
Right: Fluorescent photomicrograph of cells attached to a
sulfur crystal. Source.

When cells divide, it’s really important that they do so equitably so you don’t end up with some having too much and some too little cell material; the results of either could be deleterious. Therefore, over the billennia that life has been around, mechanisms to carefully divvy up intracellular goodies like DNA and cytoplasm have evolved. You can imagine that interest peaked in those who pay attention to such things when it was found that a group of microbes belonging to the Crenarchaeota lack any of the known cell division machinery. Crenarchaeota are a kind of Archaea that includes species known to survive in extremely hot or cold environments. And when we say extreme, we mean deep arctic-cold and volcano-hot—literally. Other known forms of life have some variant of actin (known as MreB in bacteria) or tubulin (aka FtsZ), which are involved in cell division or maintenance of cell shape. Until late 2008, when a pair of papers was published, no such proteins were found in the crenarchaea and their division remained quite mysterious.

Regardless, these hardy little archaea are getting the job done, one way or the other. It has been known for some time that the Crenarchaeota possess homologs of a system called ESCRT (endosomal sorting complex required for transport). This is an attractive candidate for managing division in these organisms. Why? Because the ESCRT system helps detach daughter from mother cell during cell division in metazoans—such as insects, octopuses, and elephants. So it seemed plausible that these proteins might be helping here as well.

On the Continuity of Biological Membranes

by Franklin M. Harold

The complex world of cell membranes. Source.

Thirty years ago, Günter Blobel of the Rockefeller University published a short paper entitled Intracellular Protein Topogenesis, which laid the conceptual foundations for our understanding of how cells build membranes. To serve their functions, peripheral and integral proteins must be inserted into the right membrane with the correct orientation, and most of the article focused on the manner in which this may be achieved. But it also underscored two startling implications of the proposed procedure: first, that every membrane must be derived from a pre-existing membrane; and second, that all extant biological membranes are descendants of the plasma membrane of the first primordial cell.

Blobel’s article became a classic, and spawned a small industry concerned with the molecular mechanisms that target proteins to the recipient membrane and then either translocate or insert them. In a nutshell, the information that specifies a nascent protein’s disposition is contained in its sequence. One segment of that sequence recognizes a receptor protein embedded in the target membrane, commonly part of the translocon; other segments specify whether the amino acid chain is to be taken clear across the membrane or inserted, and with what orientation. Membrane proteins may be processed concurrently with their translation, or after their production is complete. In prokaryotic cells the proteins are produced and handled directly; in eukaryotic cells they are first inserted into the membrane of the endoplasmic reticulum, and then transferred to their target membrane by cargo vesicles. The details can be found in textbooks of molecular cell biology. What concerns us here is the inference that membrane heredity is a fundamental principle of biology. A functional membrane, studded with a particular set of enzymes, transport carriers and receptors, can never be generated de novo; it must arise from a pre-existing membrane, either by modification (for example, the membranes that surround bacterial spores) or else by growth and division or vesiculation. Moreover, since proteins will only be inserted after interaction with a complementary receptor (and that includes the receptor protein itself), a growing “genetic” membrane propagates its own kind.

The idea that membranes are inherited was by no means novel in 1980; cytologists had been musing on it for two decades. But it was quite another matter to assert that it must be so, that “omnis membrana e membrana.”

Mother’s Love

by Elio

Binary fission is a most impressive invention. In one fell swoop, it ensures that progeny cells are born alike and endowed with the same potential for growth and survival. Simple as it sounds, it must have taken considerable evolutionary contortions to make it function so well throughout the living world. But there are cells that have adopted an alternative mechanism, where cell division is asymmetrical, where one progeny cell is made from a “mother cell” that keeps generating “babies.” The best known example is, of course, budding in yeast. But other cells also arise in this fashion, including some bacteria, the sexual spores of mushrooms, and even some plant cells.

The polarisome in a forming yeast bud. Actin cables
growing out from the polarisome transport protein
aggregates to the mother cell. Source.

So, is there an advantage to bypassing binary fission and budding instead? It would seem that way. Recent work from Tom Nyström’s lab has shown that proteins that become damaged in the course of cell growth flow back into the mother cell and leave the young bud free of such impediments. The damage to proteins is often due to oxidation by reactive oxygen species. Damaged proteins tend to form aggregates. This can be bad, getting rid of them is good. How do these proteins accumulate in the mother cell? Interestingly, protein aggregates hitch a ride on actin filaments that grow from the growing bud to the mother cell. Such filaments are assembled at the tip of the bud in a structure the authors call a “polarisome,” which is made up of core proteins plus some involved in actin polymerization (formins). Also required is the age-retardant deacetylase, a protein called Sir2 and aka sirtuin. Previously known as a life span modulator—not just in yeast but in worms, fish, and mammals as well—Sir2 has now been found to also be involved in actin-related processes, hence in polarisome formation. It gets more complicated. For an overview, we suggest a commentary by Guarente.

(A) Exponentially growing young yeast cells to compare their size and
morphology to that of old cells. (B) A typical lifespan determination.
The number of budding cycles that each of a set of 50 ‘virgin’ cells
undergoes before it stops dividing was determined by micromanipu-
lation and counting of budding cycles. (C) M is the terminal mother cell
after 15 cell cycles. Note the enormous size as compared to young cells
and the surface changes. D14 is the second but last daughter that did
not completely separate from the mother and did not give rise to new
living cells. Source.

Now let’s look at this story in a broader context. It’s not just about shipping dirty laundry off to mother. One consequence of the asymmetry of budding is that the mother cell retains its bodily integrity bud after bud, whereas this is lost when a cell divides by binary fission. In yeast, a mother cell can bud some 15-30 times, after which it conks out. How do we know? Counting the number of times a cell gives off buds is done patiently under the microscope, using a micromanipulator to remove each daughter cell once it separates from the mother cell. Keep this up until the mother cell gives off no more buds. Imagine teasing the new cells away for the mother cell 30 times or so! (This seems to have been done first in 1950 by A. A. Barton, working for a British brewing company.) This phenomenon is called senescence, and is visually illustrated by wrinkles of old age and the unusually large size of the Grande Dame. Newly formed cells start the process anew, each daughter cell becoming a mother cell of its own. However, in time, the newly made cells are less competent for further budding. Not surprisingly, yeast is a favorite for studies of cell polarization and its possible role in senescence. Many papers have been written on this subject.

A favorite connection between yeast budding and aging relies on a 120-year-old theory by August Weismann. He postulated that aging evolved from the need to separate germ cells from somatic cells. Germ cells need to be protected from damage; somatic cells can ”take it.” One of the reasons given is that additional resources must be bestowed on germ cells to ensure their genetic stability. Somatic cells, in contrast, do not have such mechanisms and thus accumulate damage.

This is one way to think about asymmetric cell division. Formally speaking, the mother cell acts as a somatic cell that produces multiple germ cells, the buds. Each, when grown, becomes a mother cell with full reproductive potential, able to produce a full complement of buds of her own. During budding, the young bud escapes from the cell damage represented by the aggregated proteins, thus foiling at least that aspect of cell aging.

If asymmetric cell division affords such protection of the germ line, why don't all cells do this? The question is not terribly relevant to multicellular somatic cells, given that they are not involved in germ line propagation (unless some investigator teases their nuclei out and introduces them into eggs). But how come more unicellular microbes haven't adopted the budding strategy? That is a question for another time.

Given that yeast is the best known of all eukaryotic organisms, allowing endless kinds of genetic manipulations, it’s no wonder that it has become a model for the study of aging. And here I thought that I would be a good subject for researching what happens in old age!

Addendum: As Qetzal noted (see his comment below), I could have mentioned that a possibly analogous phenomenon has been reported for bacteria. When E. coli divides, the "old" cell pole accumulates chaperones involved in the aggregation of (presumably) damaged proteins. Eventually the "old" cells lose their reproductive capability. Something analogous takes place in Caulobacter crescentus. Thus, bacteria may also use the strategy of segregating damaged proteins in aging cells, to the advantage of the population as a whole.

Liu, B., Larsson, L., Caballero, A., Hao, X., Öling, D., Grantham, J., & Nyström, T. (2010). The Polarisome Is Required for Segregation and Retrograde Transport of Protein Aggregates Cell, 140 (2), 257-267 DOI: 10.1016/j.cell.2009.12.031

The Limitations of LB Medium

by Hiroshi Nikaido

LB medium, also known incorrectly as Luria-Bertani medium, is widely used to grow bacterial cultures, mainly because it is easy to prepare and provides a broad base of nutrients. LB broth contains, per ml, 10 mg tryptone (a mixture of peptides formed by the digestion of casein with the pancreatic enzyme, trypsin), 5 mg yeast extract (an autolysate of yeast cells), and 5 or 10 mg NaCl. It was formulated by Giuseppe Bertani in 1951 for studying lysogeny in Escherichia coli. He called it “Lysogeny Broth,” or LB. Notably, the original formulation included 1 mg/ml glucose, which has been dropped in more recent times. This medium was designed for work at low bacterial densities, a key point of this article.

While this may appear a tasty dish for many bacteria of research interest, it is an inappropriate choice for physiological studies wherein reproducibility is required. Since only bacterial cultures in balanced growth (achieved by sufficient time in exponential growth) have a reproducible average cell size and chemical composition, none of the components of liquid media should become exhausted during growth of the culture. Is this the case with LB broth? To answer this question, we must know what limits bacterial growth in LB broth.

Getting a Handle on Cell Organization

by Franklin M. Harold

Mitotic spindle of a human cell: microtubules in green,
chromosomes in blue, centrosomes in red. Source.

Structural organization is one of the most conspicuous features of cells, and possibly the most elusive. No one really doubts that that cell functions commonly require that the right molecules be in the right place at the right time; or that spatial organization is what distinguishes a living cell from a soup of its molecular constituents. But the tradition that has dominated biological research for the past century mandates a focus on the molecules, and so our first step is commonly to grind the exquisite architecture of the living cell into a pulp. Few molecular scientists have asked whether anything irretrievable is lost by this brutal routine. Such questions as how molecules find their proper place in a framework orders of magnitude larger, or how spatial order is transmitted from one generation to the next, have been largely neglected until recently.

Two current and quite excellent short reviews afford an entry into the wilderness. Eric Karsenti takes an historical approach to the role of self-organization in creating order on the cellular scale. The physical principles are arcane, but some aspects are actually quite familiar. We have known for half a century that supra-molecular complexes often arise by self-assembly, without any input of either information or energy; examples include lipid bilayer membranes, ribosomes, microtubules, S-layers and virus particles. But the scope of self-organization has been greatly enlarged in recent years by the discovery that an array of dynamic structures can be generated in the presence of an energy source, usually ATP or GTP. The mitotic spindle of eukaryotes has been identified as a self-organizing machine, the endomembrane system may be another. Like self-assembly, self-construction (my term) requires no external source of information, but it does entail continual energy consumption. In a complementary article, Allen Liu and Daniel Fletcher survey a selection of efforts to reconstitute cellular functions in simplified systems, starting with cell-free extracts or purified proteins. Ingenious experimenters have managed to reconstitute the essentials of actin-based motility, membrane protrusion, the oscillatory system that localizes the midpoint of bacterial cells, and now also the contraction of the Z-ring. Though much remains to be learned, it is safe to conclude that the lower levels of cellular order, at least, are products of pure chemistry: they arise by interactions among the molecular constituents in ways that require the cell as a whole to supply energy and a permissive environment, but no spatial instructions.

This is excellent science, which takes us some way towards bridging the gulf between nanometer-sized molecules and cells in the range from micrometers to millimeters. It also extends the genome’s reach deep into cellular structure. In a self-organizing system, the “instructions” must be wholly inherent in the molecular parts, and ultimately derive from the corresponding genes. It is the genome that specifies the architecture of the mitotic spindle, not explicitly but indirectly: the form and even functions of the spindle are implied in the structure of the spindle proteins, and in their interactions. And if the spindle can be envisaged as a creature of self-organization, why not the entire cell? Yes, indeed─but as we ascend the hierarchy of biological organization, the meaning assigned to self-organization and its underlying mechanisms undergo significant changes. Cells do not construct themselves from pre-fabricated standard parts; instead, they grow. And that mode of self-organization is not purely chemical, for it must produce parts that have biological functions, performed in the service of a larger entity that can compete and thrive in the wide world (discussed here).

Mycobacteria Make Spores?

by Peter Setlow

The title page of Robert Koch’s original article,
The Etiology of Tuberculosis, published in the
Berliner Klinische Wochenschrift, April 10, 1882.

Rarely does news come along that threatens to overturn our cherished beliefs. Since early after the discovery of tubercle bacilli by Robert Koch in 1882, the mycobacteria have been considered not to be spore formers. Now this is being challenged by a new report.

The genus Mycobacterium encompasses a number of major human pathogens including M. tuberculosis, among the most common pathogens carried by humans. A hallmark of M. tuberculosis infections is the ability of the organism to persist asymptomatically for long periods in the host, and then reappear and cause disease. The new paper by Ghosh et al. from Uppsala University in Sweden claims that members of some Mycobacterium species form spores that resemble those produced by well-studied Bacillus species. The authors worked primarily with M. marinum, a relatively fast-growing mycobacterium with a doubling time of 4-6 hours. In old cultures they observed forms that resembled spores not only in their morphology and their resistance, but also in their accumulation of dipicolinic acid, a signature molecule associated with spores of Bacillus species. Putative spores were also observed in old M. bovis cultures. In addition, they found that Mycobacterium genomes, including that of M. tuberculosis, contain homologs of genes important for sporulation of the model spore former, Bacillus subtilis. At least some of these genes were transcribed only in old M. marinum cultures.

The Microbe That Could Be Seen

by Elio

The Microbe is so very small
You cannot make him out at all,
But many sanguine people hope
To see him through a microscope.

            From The Microbe by Hilaire Belloc, 1900

One epulo with four paramecia. Courtesy of
Esther Angert.

Biology is replete with definitions that have exceptions. A microbe (from the Greek roots mikros and bios for small life) is typically defined as a microscopic living organism invisible to the naked eye. It is clearly in the nature of living things to stretch our definitions, bend the rules. There are bacteria (surely microbes) that pooh-pooh the notion that bacteria must be small. They are large enough to be readily seen with the naked eye. But this was not appreciated for a long time; extreme bacterial gigantism is a new subject that is stretching our understanding of bacterial structure and good manners. (For gigantism among animals at the bottom of the sea, view this lovely movie.)

The current champion microbial leviathans are indisputably the cigar-shaped Epulopiscium fishelsoni that can exceed 600 μm in length, and the spherical Thiomargarita namibiensis more than 700 μm in diameter. In truth, T. namibiensis is something of an impostor, being just a thin layer of cytoplasm surrounding a colossal vacuole. Epulopiscium, on the other hand, is a genuine, cytoplasm-filled giant.

There is no "Z" in "Division"

We translate and offer a post from the Spanish blogs Curiosidades de la Microbiología (Curiosities of the Microbiological World) and Esos Pequeños Bichitos (Those Small Bugs).

by Manuel Sánchez and Miguel Vicente

Phylogenetic distribution of FtsZ protein. This protein is
essential for many Bacteria, Archaea, and organelles.

From wheels to turbines, humans have invented a goodly number of machines to perform various mechanical tasks. The same function, e.g., to move a car, can be carried out by motors of different designs. This is also true for cells, where the same function can be carried out by differently designed machines. Just as most cars use a gasoline engine, most microbes and some organelles (e.g., chloroplasts) use the same component, the protein FtsZ, to carry out cell division.

Nevertheless, just as there are cars that use an electric motor and locomotives with a steam engine, some microbes get by without FtsZ and use another system of proteins for their division.

Complex Simplicity

We translate and offer a post from the Spanish blog Esos Pequeños Bichitos (Those Small Bugs).

Bernini’s baldachin (aka canopy of state)
in the Vatican’s Basilica of Saint Peter.
The helical columns remind us of the
helical rails that transport proteins along
the membrane of bacterial cells. Credit:
Thiago.

by Miguel Vicente

The textbooks we used in the 20th century described bacterial division with one term, binary fission, implying a simple process. Nothing could be farther from the truth. Today we know that more than a dozen proteins are employed to make the division septum in Escherichia coli. They assemble in a complex sequence and all are needed. In the absence of any one, subsequent assembly is not possible and division cannot take place. Before this machinery can be assembled, an initiator protein, FtsZ, becomes localized in the middle of the cell, where it makes a constriction ring.

The Search for the Center

There are at least two mechanisms that ensure the precise location of FtsZ. One is that this protein does not function in the vicinity of the nucleoid (termed “nucleoid occlusion” by Conrad Woldringh). The other way is independent of the nucleoid and relies on the local concentration of an FtsZ inhibitor called MinC (a mechanism proposed by Larry Rothfield).