The cell wall is an essential structure for virtually all bacteria, forming a tough outer shell that protects the cell from damage and osmotic lysis. It is the target of our best antibiotics. L-form strains are wall-deficient derivatives of common bacteria that have been studied for decades. However, they are difficult to generate and typically require growth for many generations on osmotically protective media with antibiotics or enzymes that kill walled forms. Despite their potential importance for understanding antibiotic resistance and pathogenesis, little is known about their basic cell biology or their means of propagation. We have developed a controllable system for generating L-forms in the highly tractable model bacterium Bacillus subtilis. Here, using genome sequencing, we identify a single point mutation that predisposes cells to grow without a wall. We show that propagation of L-forms does not require the normal FtsZ-dependent division machine but occurs by a remarkable extrusion-resolution mechanism. This novel form of propagation provides insights into how early forms of cellular life may have proliferated.

When Pseudomonas putida KT2440 cells encounter toluene in the growth medium, they perceive it simultaneously as a potential nutrient to be metabolized, as a membrane-damaging toxic drug to be extruded, and as a macromolecule-disrupting agent from which to protect proteins. Each of these inputs requires a dedicated transcriptional response that involves a large number of genes. We used DNA array technology to decipher the interplay between these responses in P. putida KT2440 subjected to a short challenge (15 min) with toluene. We then compared the results with those in cells exposed to o-xylene (a non-biodegradable toluene counterpart) and 3-methylbenzoate (a specific substrate of the lower TOL pathway of the P. putida pWW0 plasmid). The resulting expression profiles suggest that the bulk of the available transcriptional machinery is reassigned to endure general stress, whereas only a small share of the available machinery is redirected to the degradation of the aromatic compounds. Specifically, both toluene and o-xylene induce the TOL pathways and a dedicated but not always productive metabolic program. Similarly, 3-methylbenzoate induces the expression not only of the lower meta pathway but also of the non-productive and potentially deleterious genes for the metabolism of (nonsubstituted) benzoate. In addition, toluene (and to a lesser extent o-xylene) inhibit motility functions as an unequivocal response to aromatic toxicity. We argue that toluene is sensed by P. putida KT2440 as a stressor rather than as a nutrient and that the inhibition by the aromatic compounds of many functions we tested is the tradeoff for activating stress tolerance genes at a minimal cost in terms of energy.

Cell morphogenesis in most bacteria is governed by spatiotemporal growth regulation of the peptidoglycan cell wall layer. Much is known about peptidoglycan synthesis but regulation of its turnover by hydrolytic enzymes is much less well understood. Bacillus subtilis has a multitude of such enzymes. Two of the best characterized are CwlO and LytE: cells lacking both enzymes have a lethal block in cell elongation. Here we show that activity of CwlO is regulated by an ABC transporter, FtsEX, which is required for cell elongation, unlike cell division as in Escherichia coli. Actin-like MreB proteins are thought to play a key role in orchestrating cell wall morphogenesis. B. subtilis has three MreB isologues with partially differentiated functions. We now show that the three MreB isologues have differential roles in regulation of the CwlO and LytE systems and that autolysins control different aspects of cell morphogenesis. The results add major autolytic activities to the growing list of functions controlled by MreB isologues in bacteria and provide new insights into the different specialized functions of essential cell wall autolysins.

SummaryThe peptidoglycan (PG) cell wall is a defining feature of the bacterial lineage and an important target for antibiotics, such as β-lactams and glycopeptides. Nevertheless, many bacteria are capable of switching into a cell-wall-deficient state, called the “L-form” [1–3]. These variants have been classically identified as antibiotic-resistant forms in association with a wide range of infectious diseases [4]. L-forms become completely independent of the normally essential FtsZ cell division machinery [3, 5]. Instead, L-form proliferation is driven by a simple biophysical process based on an increased ratio of surface area to cell volume synthesis [6, 7]. We recently showed that only two genetic changes are needed for the L-form transition in Bacillus subtilis [7]. Class 1 mutations work to generate excess membrane synthesis [7]. Until now, the function of the class 2 mutations was unclear. We now show that these mutations work by counteracting an increase in the cellular levels of reactive oxygen species (ROS) originating from the electron transport pathway, which occurs in wall-deficient cells. Consistent with this, addition of a ROS scavenger or anaerobic culture conditions also worked to promote L-form growth without the class 2 mutations in both Gram-positive B. subtilis and Gram-negative Escherichia coli. Our results suggest that physiological compensation for the metabolic imbalance that occurs when cell wall synthesis is blocked is crucial for L-form proliferation in a wide range of bacteria and also provide new insights into the mode of action of antibiotics that target the bacterial cell wall.

The cell wall is a defining structural feature of the bacterial subkingdom. However, most bacteria are capable of mutating into a cell-wall-deficient "L-form" state, requiring remarkable physiological and structural adaptations. L-forms proliferate by an unusual membrane deformation and scission process that is independent of the conserved and normally essential FtsZ based division machinery, and which may provide a model for the replication of primitive cells. Candidate gene screening revealed no requirement for the cytoskeletal systems that might actively drive membrane deformation or scission. Instead, we uncovered a crucial role for branched-chain fatty acid (BCFA) synthesis. BCFA-deficient mutants grow and undergo pulsating shape changes, but membrane scission fails, abolishing the separation of progeny cells. The failure in scission is associated with a reduction in membrane fluidity. The results identify a step in L-form proliferation and demonstrate that purely biophysical processes may have been sufficient for proliferation of primitive cells.

SummaryL-forms are variants of common bacteria that can grow and proliferate without a cell wall. Little is known about their molecular cell biology but they undergo a remarkable mode of proliferation that is independent of the normally essential FtsZdependent division machinery. We have isolated a strain of Bacillus subtilis that can quickly and quantitatively convert from the walled to the L-form state. Analysis of the transition process identified an unexpected 'escape' step needed for L-form emergence from the rod. Mutations in two different genes, walR and sepF, contribute to the high frequency of escape: walR, a transcriptional regulator involved in cell wall homeostasis; and sepF, required for accurate and efficient cell division. Time-lapse imaging shows that the mutations act by facilitating the release of the L-form from its walled parent cell but that they act in different ways. The walR mutation renders the activity of the protein partially constitutive, inappropriately upregulating the activity of autolytic enzymes that weaken the cell wall. The sepF mutation probably works by perturbing the formation of a properly constructed division septum, generating a mechanical breach in the wall. The new strain provides a powerful experimental system for studying the genetics and cell biology of L-forms.

b L-forms are cell wall-deficient bacteria that can grow and proliferate in osmotically stabilizing media. Recently, a strain of the Gram-positive model bacterium Bacillus subtilis was constructed that allowed controlled switching between rod-shaped wildtype cells and corresponding L-forms. Both states can be stably maintained under suitable culture conditions. Because of the absence of a cell wall, L-forms are known to be insensitive to ␤-lactam antibiotics, but reports on the susceptibility of L-forms to other antibiotics that interfere with membrane-anchored steps of cell wall biosynthesis are sparse, conflicting, and strongly influenced by strain background and method of L-form generation. Here we investigated the response of B. subtilis to the presence of cell envelope antibiotics, with regard to both antibiotic resistance and the induction of the known LiaRS-and BceRS-dependent cell envelope stress biosensors. Our results show that B. subtilis L-forms are resistant to antibiotics that interfere with the bactoprenol cycle, such as bacitracin, vancomycin, and mersacidin, but are hypersensitive to nisin and daptomycin, which both affect membrane integrity. Moreover, we established a lacZ-based reporter gene assay for L-forms and provide evidence that LiaRS senses its inducers indirectly (damage sensing), while the Bce module detects its inducers directly (drug sensing).

Pseudomonas putida encodes 20 extracytoplasmic sigma factors (ECFs). In this study, we show that one of these ECFs, known as ECF-Pp12 (PP3006), plays a role in tolerance of toluene and other organic solvents. Based on this finding, we have called the gene that encodes this new ECF rpoT. The rpoT gene forms an operon with the preceding gene and with the gene located downstream. The translated gene product of the open reading frame PP3005 is an inner membrane protein, whereas the PP3007 protein is periplasmic. A nonpolar ⌬rpoT mutant was generated by homologous recombination, and survival of the mutant was tested under various stress conditions. The mutant strain was hypersensitive to toluene and other solvents but just as tolerant as the wild type of stress imposed by heat, antibiotics, NaCl, paraquat, sodium dodecyl sulfate, H 2 O 2 , and benzoate. In the ⌬rpoT mutant background, expression of around 50 transcriptional units was affected: 31 cistrons were upregulated, and 23 cistrons were downregulated. This indicates that about 1% of all P. putida genes are under the direct or indirect influence of RpoT. The rpoT gene controls the expression of a number of membrane proteins, including components of the respiratory chains, porins, transporters, and multidrug efflux pumps. Hypersensitivity of the P. putida RpoT-deficient mutant to organic solvents can be attributed to the fact that in the ⌬rpoT strain, expression of the toluene efflux pump ttgGHI genes is severalfold lower than in the parental strain.In natural environments, microorganisms are exposed to changing conditions and have therefore developed a series of strategies to cope with these stressors (17, 48). With world industrialization, humans have synthesized a large number of chemicals, many of which reach the biosphere and constitute a new burden for the environment. Among the most toxic chemicals are organic solvents, such as toluene, xylenes, and styrene, which dissolve in the cell membrane, disorganize it, cause the loss of lipids and proteins, and eventually lead to cell death (8,45,56). Microorganisms have developed various mechanisms to resist the lethal effects of these chemicals. One of the most relevant of these mechanisms is the active reduction of their entry into the cells through the action of membrane efflux pumps, which belong to the group of multidrug resistance pumps (45). These efflux pumps extrude a broad range of structurally unrelated synthetic and natural chemicals and thus constitute an effective barrier against toxic chemicals.Pseudomonas putida DOT-T1E exhibits the unusual property of being highly tolerant of organic solvents (39, 47). Solvent tolerance in this strain is ultimately the result of an interplay between three efflux systems known as TtgABC, TtgDEF, and TtgGHI, which have been assigned to the root nodulation cell division family (15,45,46,55). Of these three efflux pumps, the TtgGHI pump seems to be the most critical for the removal of solvents from a quantitative point of view (51). The ttgGHI and the ttgDEF operons ...