Bacterial Cell Morphogenesis Does Not Require a Preexisting Template Structure

SummaryMorphogenesis, the development of shape or form in cells or organisms, is a fundamental but poorly understood process throughout biology. In the bacterial domain, cells have a wide range of characteristic shapes, including rods, cocci, and spirals. The cell wall, composed of a simple meshwork of long glycan strands crosslinked by short peptides (peptidoglycan, PG) and anionic cell wall polymers such as wall teichoic acids (WTAs), is the major determinant of cell shape. It has long been debated whether the formation of new wall material or the transmission of shape from parent to daughter cells requires existing wall material as a template [1–3]. However, rigorous testing of this hypothesis has been problematical because the cell wall is normally an essential structure. L-forms are wall-deficient variants of common bacteria that have been classically identified as antibiotic-resistant variants in association with a wide range of infectious diseases [4–6]. We recently determined the genetic basis for the L-form transition in the rod-shaped bacterium Bacillus subtilis and thus how to generate L-forms reliably and reproducibly [7, 8]. Using the new L-form system, we show here that we can delete essential genes for cell wall synthesis and propagate cells in the long-term absence of a cell wall template molecule. Following genetic restoration of cell wall synthesis, we show that the ability to generate a classical rod-shaped cell is restored, conclusively rejecting template-directed models, at least for the establishment of cell shape in B. subtilis

Domain Movement within a Gene: A Novel Evolutionary Mechanism for Protein Diversification

A protein function is carried out by a specific domain localized at a specific position. In the present study, we report that, within a gene, a specific amino acid sequence can move between a certain position and another position. This was discovered when the sequences of restriction-modification systems within the bacterial species Helicobacter pylori were compared. In the specificity subunit of Type I restriction-modification systems, DNA sequence recognition is mediated by target recognition domain 1 (TRD1) and TRD2. To our surprise, several sequences are shared by TRD1 and TRD2 of genes (alleles) at the same locus (chromosomal location); these domains appear to have moved between the two positions. The gene/protein organization can be represented as x-(TRD1)-y-x-(TRD2)-y, where x and y represent repeat sequences. Movement probably occurs by recombination at these flanking DNA repeats. In accordance with this hypothesis, recombination at these repeats also appears to decrease two TRDs into one TRD or increase these two TRDs to three TRDs (TRD1-TRD2-TRD2) and to allow TRD movement between genes even at different loci. Similar movement of domains between TRD1 and TRD2 was observed for the specificity subunit of a Type IIG restriction enzyme. Similar movement of domain between TRD1 and TRD2 was observed for Type I restriction-modification enzyme specificity genes in two more eubacterial species, Streptococcus pyogenes and Mycoplasma agalactiae. Lateral domain movements within a protein, which we have designated DOMO (domain movement), represent novel routes for the diversification of proteins

Dissecting the roles of peptidoglycan synthetic and autolytic activities in the walled to L-form bacterial transition

Bacterial cells are surrounded by a peptidoglycan (PG) wall, which is a crucial target for antibiotics. It is well known that treatment with cell wall-active antibiotics occasionally converts bacteria to a non-walled “L-form” state that requires the loss of cell wall integrity. L-forms may have an important role in antibiotic resistance and recurrent infection. Recent work has revealed that inhibition of de novo PG precursor synthesis efficiently induces the L-form conversion in a wide range of bacteria, but the molecular mechanisms remain poorly understood. Growth of walled bacteria requires the orderly expansion of the PG layer, which involves the concerted action not just of synthases but also degradative enzymes called autolysins. Most rod-shaped bacteria have two complementary systems for PG insertion, the Rod and aPBP systems. Bacillus subtilis has two major autolysins, called LytE and CwlO, which are thought to have partially redundant functions. We have dissected the functions of autolysins, relative to the Rod and aPBP systems, during the switch to L-form state. Our results suggest that when de novo PG precursor synthesis is inhibited, residual PG synthesis occurs specifically via the aPBP pathway, and that this is required for continued autolytic activity by LytE/CwlO, resulting in cell bulging and efficient L-form emergence. The failure of L-form generation in cells lacking aPBPs was rescued by enhancing the Rod system and in this case, emergence specifically required LytE but was not associated with cell bulging. Our results suggest that two distinct pathways of L-form emergence exist depending on whether PG synthesis is being supported by the aPBP or RodA PG synthases. This work provides new insights into mechanisms of L-form generation, and specialisation in the roles of essential autolysins in relation to the recently recognised dual PG synthetic systems of bacteria

Cell growth of wall-free L-form bacteria is limited by oxidative damage

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

Butyltin and phenyltin residues in water, sediment and biological samples collected from Otsuchi Bay, Japan

Between 1996-2001, butyltin (BT) and phenyltin (PT) compounds were monitored in water, sediment, plankton mussels and fish from Otsuchi Bay. The changes of tributyltin (TBT) compounds in water and sediment were not observed during study period, however TBTs in plankton, mussels between 1997-1999 decreased in comparison with those in 1996. The current status of BTs and PTs in Otsuchi Bay was investigated. TBT in water, sediment, plankton, mussels and fish ranged of , 0.016-0.110mgkg^ dry, 0.010-0.255mgkg^ dry, 0.012-0.048mgkg^ wet and 0.009-0.029mgkg^ wet, respectively. TBT concentrations were high near the shipyard. Triphenyltin (TPT) compounds in water and mussel were not detected. TPT in sediment, plankton and fish ranged of dry, dry and wet, respectively

Distribution of Stable DnaA-Binding Sites on the Bacillus Subtilis Genome Detected using a Modified ChIP-chip Method

We developed a modified ChIP-chip method, designated ChAP-chip (Chromatin Affinity Precipitation coupled with tiling chip). The binding sites of Bacillus subtilis Spo0J determined using this technique were consistent with previous findings. A DNA replication initiator protein, DnaA, formed stable complexes at eight intergenic regions on the B. subtilis genome. Characterization of the binding sequences suggested that two factors—the local density of DnaA boxes and their affinities for DnaA—are critical for stable binding. We further showed that in addition to autoregulation, DnaA directly modulate the expression of sda in a positive, and ywlC and yydA in a negative manner. Examination of possible stable DnaA-binding sequences in other Bacillus species suggested that DnaA-dependent regulation of those genes is maintained in most bacteria examined, supporting their biological significance. In addition, a possible stable DnaA-binding site downstream of gcp is also suggested to be conserved. Furthermore, potential DnaA-binding sequences specific for each bacterium have been identified, generally in close proximity to oriC. These findings suggest that DnaA plays several additional roles, such as control of the level of effective initiator, ATP-DnaA, and/or stabilization of the domain structure of the genome around oriC for the proper initiation of chromosome replication

Azuki Bean Juice Lowers Serum Triglyceride Concentrations in Healthy Young Women

Effects of azuki bean juice supplementation, prescribed according to a Kanpo medicine regimen, on serum lipid concentrations were studied. Healthy young Japanese women were recruited and were randomly assigned to one of the three groups using a parallel-group design. Control (n = 10), azuki (n = 11) and Concentrated azuki (CA) (n = 12) juice groups consumed 150 g daily of the isocaloric assigned juice for one menstrual cycle with their usual diet. Triglyceride concentrations were decreased in the azuki juice group (p<0.05) and tended to be decreased in the CA juice group (p = 0.055). Triglyceride concentrations in the azuki and CA juice groups decreased by 0.170 mmol/liter (15.4%) and 0.159 mmol/liter (17.9%), respectively (p<0.05). The azuki and CA juice used in this study inhibited pancreatic lipase activity 29.2% and 56.9%, respectively, in vitro. Lipid peroxide changes, based on ANCOVA with the initial level and α-tocopherol changes as covariates, did not differ among the three groups. Serum low density lipoprotein-cholesterol and high density lipoprotein-cholesterol (HDL) cholesterol concentrations did not change. Thus, azuki bean juice intake, as a traditional Kampo prescription, might be beneficial for preventing hypertriglyceridemia

Unraveling the Nature of Unidentified High Galactic Latitude Fermi/LAT Gamma-ray Sources with Suzaku

We report on the results of deep X-ray follow-up observations of four unidentified Fermi/LAT gamma-ray sources at high Galactic latitudes using Suzaku. The studied objects were detected with high significance during the first 3 months of Fermi/LAT operation, and subsequently better localized in the Fermi/LAT 1 year catalog (1FGL). Possible associations with pulsars and active galaxies have subsequently been discussed, and our observations provide an important contribution to this debate. In particular, an X-ray point source was found within the 95% confidence error circle of 1FGL J1231.1-1410. X-ray spectrum is well-fitted by a blackbody with an additional power-law. This supports the recently claimed identification of this source with a millisecond pulsar (MSP) PSR J1231-1411. Concerning 1FGL J1311.7-3429, two X-ray sources were found within the LAT error circle. Even though the X-ray spectral and variability properties were accessed, their nature and relationship with the gamma-ray source remain uncertain. We found several weak X-ray sources in the field of 1FGL J1333.2+5056, one coinciding with CLASS J1333+5057. We argue the available data are consistent with the association between these two objects. Finally, we have detected an X-ray source in the vicinity of 1FGL J2017.3+0603. This object was recently suggested to be associated with a newly discovered MSP PSR J2017+0603, because of the spatial-coincidence and the gamma-ray pulse detection. We have only detected the X-ray counterpart of the CLASS J2017+0603, while we determined an X-ray flux upper limit at the pulsar position. All in all, our studies indicate while a significant fraction of unidentified high Galactic latitude gamma-ray sources is related to the pulsar and blazar phenomena, associations with other classes of astrophysical objects are still valid options.Comment: Accepted for publication in the Ap

Author Correction: Possible role of L-form switching in recurrent urinary tract infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper