Interplay of the Serine/Threonine-Kinase StkP and the Paralogs DivIVA and GpsB in Pneumococcal Cell Elongation and Division

Despite years of intensive research, much remains to be discovered to understand the regulatory networks coordinating bacterial cell growth and division. The mechanisms by which Streptococcus pneumoniae achieves its characteristic ellipsoid-cell shape remain largely unknown. In this study, we analyzed the interplay of the cell division paralogs DivIVA and GpsB with the ser/thr kinase StkP. We observed that the deletion of divIVA hindered cell elongation and resulted in cell shortening and rounding. By contrast, the absence of GpsB resulted in hampered cell division and triggered cell elongation. Remarkably, ΔgpsB elongated cells exhibited a helical FtsZ pattern instead of a Z-ring, accompanied by helical patterns for DivIVA and peptidoglycan synthesis. Strikingly, divIVA deletion suppressed the elongated phenotype of ΔgpsB cells. These data suggest that DivIVA promotes cell elongation and that GpsB counteracts it. Analysis of protein-protein interactions revealed that GpsB and DivIVA do not interact with FtsZ but with the cell division protein EzrA, which itself interacts with FtsZ. In addition, GpsB interacts directly with DivIVA. These results are consistent with DivIVA and GpsB acting as a molecular switch to orchestrate peripheral and septal PG synthesis and connecting them with the Z-ring via EzrA. The cellular co-localization of the transpeptidases PBP2x and PBP2b as well as the lipid-flippases FtsW and RodA in ΔgpsB cells further suggest the existence of a single large PG assembly complex. Finally, we show that GpsB is required for septal localization and kinase activity of StkP, and therefore for StkP-dependent phosphorylation of DivIVA. Altogether, we propose that the StkP/DivIVA/GpsB triad finely tunes the two modes of peptidoglycan (peripheral and septal) synthesis responsible for the pneumococcal ellipsoid cell shape.     

Demonstration of Ribosomes in Mesosomes Associated with Bacillus subtilis Protoplasts

The physiological differences between Bacillus subtilis (ATCC 6633) cells derived from a glucose-salts-yeast extract (GSY) medium and those of cells from tryptose broth permitted the identification of variables in protoplasting environments which noticeably affected the clarity of mesosomal ribosomes. They were the sucrose and magnesium ion concentrations and the type of buffer used. The environment suitable for conversion of GSY cells to the protoplast state was a 0.02 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.2, containing 0.6 M sucrose and 0.03 M MgCl(2). Branched mesosomal tubules and a unique organization of vesicles were detected in thin sections and in negative stains of the specimens. Ribosomes were demonstrable in the extruded structures associated with protoplasts that had been prepared according to four fixation schedules and embedded in either of two epoxy plastics. Adjustments in the fixation schedules improved the clarity of the large bodies of protoplast cytoplasm to a degree equivalent to that of their dangling appendages.     

Proteins of ribosome-bearing and free-membrane domains in Bacillus subtilis

In lysates of Bacillus subtilis a free-membrane fraction without ribosomes can be separated from the denser membrane-ribosome complexes. As determined by one-dimensional sodium dodecyl sulfate gel electrophoresis, these two fractions differ markedly in protein composition; at least six major bands (molecular weights, 130,000, 92,000, 68,000, 64,000, 45,000, and 31,000) are essentially unique to the complexed-membrane fraction (CM proteins), and two are unique to the free-membrane fraction. After growth was slowed, the proportion of the free-membrane fraction increased, but the composition of this fraction was the same, whereas after puromycin treatment, which abruptly increased the proportion of the free-membrane fraction, this fraction contained CM proteins. Thus, it appears that the two fractions recovered from growing cells represent topographically and functionally distinct domains. In addition, the effect of growth rate suggests that formation of the complexed domain is regulated at least roughly in parallel with the formation of ribosomes. The separation of these membrane fractions should facilitate the study of protein secretion, membrane topography, and morphogenesis in bacteria.     

Overexpression of the cat-86 Gene Is Associated with Thermosensitivity in Bacillus subtilis

Bacillus subtilis harboring the cat-86 constitutive plasmid pPL708C2 with an ochre mutation at the 9th codon (terc 9) was sensitive to chloramphenicol (Cm^s) and exhibited relative thermostability when heated at 47°C. Reversion to chloramphenicol resistance (Cm^r) occurred at a frequency of 5.4 × 10⁻⁸. All of the plasmid Cm^r revertants tested were thermosensitive. Similarly, wild-type pPL708C2 present in B. subtilis also rendered the bacterium thermosensitive. When a nonsense mutation is introduced at codon 141, however, this terc 141 variant of pPL708C2 failed to thermosensitize B. subtilis. Another variant of pPL708C2 that produces intact yet catalytically inactive CAT-86 has both His-16 and His-17 at the active site replaced by Pro. Nevertheless, cells of B. subtilis carrying this variant were thermosensitive. Plasmid-free and pPL708C2-bearing strains did not exhibit differences in major heat shock proteins. Electron micrographs revealed a threefold increase of inclusion bodies present in a strain harboring pPL708C2 when compared with those in an isogenic plasmid-free strain. Received: 26 July 1999 / Accepted: 30 August 1999     

Induction and Propagation of a Bacillus subtilis L Form in Natural and Synthetic Media

The L form of Bacillus subtilis NRRL B-3275 was induced in a 7% NaCl broth medium and subsequently propagated in natural and synthetic media. The L form grew readily in tryptone broth supplemented with glucose, NaCl, and phosphate buffer, and in a synthetic medium containing only glucose and biotin, in addition to the required salts. Successive transfers from the bacillus inoculum and subsequent large bodies in the tryptone broth with 7% NaCl resulted in gradual selection or transition from the bacillary form to a stable L form without the addition of an antibiotic. The number of viable granules attained in the broth culture exceeded 9 x 10(7) per ml, and numerous large bodies were always present in rapidly growing cultures.     

Cell wall turnover in growing and nongrowing cultures of Bacillus subtilis

Cell wall turnover was studied in cultures of Bacillus subtilis in which growth was inhibited by nutrient starvation or by the addition of antibiotics. Concomitantly, the synthesis of wall, as measured by the incorporation of radioactively labeled N-acetylglucosamine, was followed in some of these cultures. In potassium- or phosphate-starved cultures, growth stopped, but wall turnover continued at a rate slightly lower than that in the control cultures. Lysis of cells did not occur. In glucose-starved cultures, continued wall turnover caused lysis of cells, since wall synthesis apparently was inhibited. The same phenomenon was observed after growth arrest by the addition of wall synthesis inhibitors such as fosfomycin, cycloserine, penicillin G, and vancomycin. Growth arrest by the addition of chloramphenicol allowed the continuation of wall synthesis; therefore, the observed turnover generally did not cause cell lysis.     

Levels of Germination Proteins in Bacillus subtilis Dormant,Superdormant, and Germinating Spores

Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiple-reaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.     

Localization in the Cell and Extraction of Alkaline Phosphatase from Bacillus subtilis

Study of protoplasts, lysed protoplasts, and cells treated with lysozyme in the absence of osmotic stabilizer suggested that the alkaline phosphatase (EC 3.1.3.1.) of Bacillus subtilis is located in the protoplasmic membrane. Cytochemical evidence in support of this view is presented. The enzyme protein was strongly bound to the membrane structure and could not be solubilized by a number of treatments known to release enzymes from membranes and other lipoprotein structures. Alkaline phosphatase was, however, solubilized by treatment of intact B. subtilis cells or isolated protoplasmic membranes with strong salt solutions at pH 7.2, suggesting that electrostatic forces are responsible for the association between membrane and enzyme protein. Dialysis of alkaline phosphatase solutions against buffer of low ionic strength resulted in precipitation of the enzyme.     

Cell wall synthesis and initiation of deoxyribonucleic acid replication in Bacillus subtilis.

We have observed a connection between cell wall synthesis and the initiation of chromosome replication in Bacillus subtilis. Initiation of chromosome replication was prevented in synchronous cultures in the presence of the cell wall synthesis inhibitor vancomycin. When vancomycin was added to the cultures after initiation of chromosome replication, one round of replication was completed but no reinitiation occurred. Similar results were obtained when cell wall synthesis was inhibited by ristocetin, cycloserine, cloxacillin, or cephaloridine. When sucrose was added to the medium, initiation of deoxyribonucleic acid replication occurred in the presence of vancomycin, to an extent which allowed replication of no more than approximately one-half of the deoxyribonucleic acid of the culture. The same was found in cultures of spheroplasts of B. subtilis. However, initiation of chromosome replication in spheroplasts was completely insensitive to cloxacillin.     

Numbers of Individual Nutrient Germinant Receptors and Other Germination Proteins in Spores of Bacillus subtilis

Germination of dormant Bacillus subtilis spores with specific nutrient germinants is dependent on a number of inner membrane (IM) proteins, including (i) the GerA, GerB, and GerK germinant receptors (GRs) that respond to nutrient germinants; (ii) the GerD protein, essential for optimal GR function; and (iii) SpoVA proteins, essential for the release of the spore-specific molecule dipicolinic acid (DPA) during spore germination. Levels of GR A and C subunit proteins, GerD, and SpoVAD in wild-type spores were determined by Western blot analysis of spore fractions or total disrupted spores by comparison with known amounts of purified proteins. Surprisingly, after disruption of decoated B. subtilis spores with lysozyme and fractionation, ∼90% of IM fatty acids and GR subunits remained with the spores'' insoluble integument fraction, indicating that yields of purified IM are low. The total lysate from disrupted wild-type spores contained ∼2,500 total GRs/spore: GerAA and GerAC subunits each at ∼1,100 molecules/spore and GerBC and GerKA subunits each at ∼700 molecules/spore. Levels of the GerBA subunit determined previously were also predicted to be ∼700 molecules/spore. These results indicate that the A/C subunit stoichiometry in GRs is most likely 1:1, with GerA being the most abundant GR. GerD and SpoVAD levels were ∼3,500 and ∼6,500 molecules/spore, respectively. These values will be helpful in formulating mathematic models of spore germination kinetics as well as setting lower limits on the size of the GR-GerD complex in the spores'' IM, termed the germinosome.     

The Role of Cell Contraction and Adhesion in Dictyostelium Motility

The crawling motion of Dictyostelium discoideum on substrata involves a number of coordinated events including cell contractions and cell protrusions. The mechanical forces exerted on the substratum during these contractions have recently been quantified using traction force experiments. Based on the results from these experiments, we present a biomechanical model of the contraction phase of Dictyostelium discoideum motility with an emphasis on the adhesive properties of the cell-substratum contact. Our model assumes that the cell contracts at a constant rate and is bound to the substratum by adhesive bridges that are modeled as elastic springs. These bridges are established at a spatially uniform rate while detachment occurs at a spatially varying, load-dependent rate. Using Monte Carlo simulations and assuming a rigid substratum, we find that the cell speed depends only weakly on the detachment kinetics of the cell-substratum interface, in agreement with experimental data. By varying the parameters that control the adhesive and contractile properties of the cell, we are able to make testable predictions. We also extend our model to include a flexible substrate and show that our model is able to produce substratum deformations and force patterns that are quantitatively and qualitatively in agreement with experimental data.     

Processing of Bacillus cereus 569/H beta-lactamase I in Escherichia coli and Bacillus subtilis

The gene for Bacillus cereus 569/H beta-lactamase I, penPC, has recently been cloned and sequenced (Mézes, P. S. F., Yang, Y. Q., Hussain, M., and Lampen, J. O. (1983) FEBS Lett. 161, 195-200). A typical prokaryotic signal peptide but with no lipoprotein modification site, as present in the Bacillus licheniformis 749/C beta-lactamase, was indicated by the DNA sequence for this secretory protein. We have here purified the beta-lactamase I products found in Escherichia coli and Bacillus subtilis carrying penPC and have determined the first 20 NH2-terminal amino acids of each of the forms. Processing of the beta-lactamase I in E. coli occurs at a single site which is characteristic for cleavage by a signal peptidase. B. subtilis secreted two distinct products to the culture medium which were both smaller than the single product formed in E. coli. Sequencing of [35S]Met-labeled pre-beta-lactamase I from phenylethyl alcohol-treated cells of B. cereus 569/H indicated that UUG is being utilized as the initiation codon for penPC. The same result was obtained for the pre-beta-lactamase I from similarly treated cells of the closely related B. cereus 5/B strain.     

Organized Cell Swimming Motions in Bacillus subtilis Colonies: Patterns of Short-Lived Whirls and Jets

The swimming motions of cells within Bacillus subtilis colonies, as well as the associated fluid flows, were analyzed from video films produced during colony growth and expansion on wet agar surfaces. Individual cells in very wet dense populations moved at rates between 76 and 116 μm/s. Swimming cells were organized into patterns of whirls, each approximately 1,000 μm², and jets of about 95 by 12 μm. Whirls and jets were short-lived, lasting only about 0.25 s. Patterns within given areas constantly repeated with a periodicity of approximately 1 s. Whirls of a given direction became disorganized and then re-formed, usually into whirls moving in the opposite direction. Pattern elements were also organized with respect to one another in the colony. Neighboring whirls usually turned in opposite directions. This correlation decreased as a function of distance between whirls. Fluid flows associated with whirls and jets were measured by observing the movement of marker latex spheres added to colonies. The average velocity of markers traveling in whirls was 19 μm/s, whereas those traveling in jets moved at 27 μm/s. The paths followed by markers were aligned with the direction of cell motion, suggesting that cells create flows moving with them into whirls and along jets. When colonies became dry, swimming motions ceased except in regions close to the periphery and in isolated islands where cells traveled in slow whirls at about 4 μm/s. The addition of water resulted in immediate though transient rapid swimming (> 80 μm/s) in characteristic whirl and jet patterns. The rate of swimming decreased to 13 μm/s within 2 min, however, as the water diffused into the agar. Organized swimming patterns were nevertheless preserved throughout this period. These findings show that cell swimming in colonies is highly organized.     

Cell cycle-dependent localization of two novel prokaryotic chromosome segregation and condensation proteins in Bacillus subtilis that interact with SMC protein

Disruption of ypuG and ypuH open reading frames in Bacillus subtilis leads to temperature-sensitive slow growth, a defect in chromosome structure and formation of anucleate cells. The genes, which were named scpA and scpB, were found to be epistatic to the smc gene. Fusions of ScpA and ScpB to the fluorescent proteins YFP or CFP showed that both proteins co-localize to two or four discrete foci that were present at mid-cell in young cells, and within both cell halves, generally adjacent to chromosomal origin regions, in older cells. ScpA and ScpB foci are associated with DNA and depend on the presence of SMC and both Scps. ScpA and ScpB are associated with each other and with SMC in vivo, as determined using the FRET technique and immunoprecipitation assays. Genes similar to scpA and scpB are present in many bacteria and archaea, which suggests that their gene products form a condensation complex with SMC in most prokaryotes. The observed foci could constitute condensation factories that pull DNA away from mid-cell into both cell halves.     

Mutagenicity screening of crude drugs with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay

This paper describes the screening studies of 104 commercial crude drugs for mutagenicity by the rec-assay with Bacillus subtilis as well as the reversion assay with Ames strains TA98 and TA100 of Salmonella typhimurium. The rec-assays showed that 13 water extracts and 27 methanol extracts of the crude drugs were positive. The Ames assays with or without metabolic activation showed that 24 water extracts and 16 methanol extracts were mutagenic. In total, mutagenic activities were found in 45 samples among the 104 crude drugs tested.     

Whole Cell Formaldehyde Cross-Linking Simplifies Purification of Mitochondrial Nucleoids and Associated Proteins Involved in Mitochondrial Gene Expression

Mitochondrial DNA/protein complexes (nucleoids) appear as discrete entities inside the mitochondrial network when observed by live-cell imaging and immunofluorescence. This somewhat trivial observation in recent years has spurred research towards isolation of these complexes and the identification of nucleoid-associated proteins. Here we show that whole cell formaldehyde crosslinking combined with affinity purification and tandem mass-spectrometry provides a simple and reproducible method to identify potential nucleoid associated proteins. The method avoids spurious mitochondrial isolation and subsequent multifarious nucleoid enrichment protocols and can be implemented to allow for label-free quantification (LFQ) by mass-spectrometry. Using expression of a Flag-tagged Twinkle helicase and appropriate controls we show that this method identifies many previously identified nucleoid associated proteins. Using LFQ to compare HEK293 cells with and without mtDNA, but both expressing Twinkle-FLAG, identifies many proteins that are reduced or absent in the absence of mtDNA. This set not only includes established mtDNA maintenance proteins but also many proteins involved in mitochondrial RNA metabolism and translation and therefore represents what can be considered an mtDNA gene expression proteome. Our data provides a very valuable resource for both basic mitochondrial researchers as well as clinical geneticists working to identify novel disease genes on the basis of exome sequence data.