Novos circuitos regulatórios de controlo espacio-temporal do desenvolvimento em bacillus subtilis

RESUMO: A esporulação em Bacillus subtilis é controlada por uma cascata de factores sigma da polimerase do RNA. F e E controlam os estágios precoces do desenvolvimento no pré-esporo e na célula mãe, respectivamente. Numa fase intermédia da diferenciação, quando a célula mãe acaba por envolver o pré-esporo, F é substituído por G e E é substituído por K. Vários mecanismos asseguram que a actividade dos diferentes factores sigma seja confinada a uma janela temporal precisa na célula adequada. Neste estudo, investigámos a função de um factor anti-G, designado por CsfB. Mostramos que para além da sua função de inibição da actividade do factor G em células pré-divisionais, CsfB é também necessário na célula mãe num estágio tardio do desenvolvimento. Mostramos que a expressão de csfB é activada na célula mãe a partir de um promotor dependente de K. Contudo, demonstramos que CsfB interage directamente com E e não com K, e que CsfB é suficiente para inibir a actividade transcricional dependente de E em células vegetativas de B. subtilis. Propomos que CsfB contribui para reduzir o período dependente de E, na linha de expressão genética da célula mãe, desse modo reduzindo a sobreposição entre os regulões E e K e aumentado a fidelidade do processo de desenvolvimento. Uma segunda proteína, YabK, partilha semelhança estrutural com CsfB. YabK é produzida no pré-esporo sob o comando de F, e é necessária para a esporulação. YabK contribui para a transição F/G no programa genético do pré-esporo, porque uma mutação que torna F sensível a CsfB ultrapassa parcialmente a função de YabK na esporulação. No entanto, YabK e CsfB funcionam por mecanismos diferentes, uma vez que YabK não liga directamente a F.---------ABSTRACT: Gene expression during spore development in Bacillus subtilis is governed by a cascade of RNA polymerase sigma factors. F and E control the early stages of development in the forespore and in the mother cell, respectively. At an intermediate stage in the differentiation process, when the larger mother cell finishes engulfment of the smaller forespore, F is replaced by G and E is replaced by K. Several mechanisms ensure the proper timing of activation of the cell type-specific sigma factors. Here, we have investigated the funtion of an anti-sigma G factor, called CsfB. We show here that in addition to its role in inhibiting G in pre-divisional cells, CsfB is also required in the mother cell at a late stage in development. We show that the expression of csfB is activated in the mother cell from a K-specific promoter. However, we demonstrate that CsfB binds directly to E but not to K in a yeast two-hybrid assay, and that CsfB is sufficient to inhibit E-dependent transcriptional activity in vegetative cells of B. subtilis. We posit that CsfB contributes to shutting off the early, E-controlled period in the mother cell line of gene expression, thus reducing the overlap between deployment of the E and K regulons and increasing the fidelity of the developmental process. A second protein, YabK, shares structural similarity with CsfB. YabK is produced in the forespore under F control, and is required for efficient sporulation. YabK contributes to the transition from the F- to the G-dependent period of gene expression, because a mutation that renders F sensitive to CsfB partially bypasses the need for YabK. Yet, YabK and CsfB must function in the control of sigma factor activity by different mechanisms because YabK does not bind directly to F.

Análise da estrutura e da função da proteína morfogenética RodZ de Bacillus subtilis

Resumo: RodZ é um componente do sistema morfogenético das células bacterianas. É uma proteína transmembranar que localiza em bandas ao longo do eixo longitudinal da célula. Em Bacillus subtilis, RodZ consiste numa porção citoplasmática, RodZn, e em uma parte extra-citoplasmática, RodZc. RodZn contém um domínio em helixturn- helix (HTH), enquanto que RodZc pode ser dividido num domínio coiled-coil e num domínio terminal C, de função desconhecida. Um segmento transmembranar (TM) único separa RodZn de RodZc. A eliminação de rodZ causa alongamento do nucleóide e leva à produção de células polares nucleadas. Aqui, mostramos que RodZn é estruturado, estável e em hélice α. Descobrimos que as substituições Y32A e L33A na suposta hélice de reconhecimento (3) do motivo HTH, bem como as substituições Y49A e F53A, fora do motivo HTH (4), causam divisão assimétrica, mas apenas as últimas levam à deslocalização sub-celular de RodZ. Sugerimos que as hélices 3 e 4 são utilizadas para uma interacção proteína-proteína ou proteína- DNA essencial para divisão celular enquanto que 4 deve contactar um componente do citosqueleto, possivelmente MreB, uma vez que a correcta localização sub-celular de RodZ depende desta proteína. Em todos os mutantes as células polares são anucleadas, pelo que concluímos que o alongamento do nucleóide não é um prérequisito para divisão assimétrica. RodZc é largamente não estruturado mas com conteúdo de folha , sendo estabilizado pelo domínio coiled-coil. Mostramos uma relação homóloga entre RodZc e a bomba de transporte Na+/Ca2+ NCX1 e identificámos dois resíduos no domínio C, G265 e N275, essenciais para a manutenção da forma celular. Estes resíduos fazem parte de um motivo em gancho que pode actuar como um local de interacção com um ligando desconhecido. RodZn e RodZc são monoméricos em solução. Contudo, na membrana, RodZ interage consigo própria num sistema de dois híbridos (Split-Ubiquitin) em levedura, sugerindo que possa formar multímeros in vivo.-----------ABSTRACT: RodZ is a transmembrane component of the bacterial core morphogenic apparatus. RodZ localizes in bands long the longitudinal axis of the cell, and it is though to functionally link the cell wall to the actin cytoskeleton. In Bacillus subtilis, RodZ consists of a cytoplasmic moiety, RodZn, and an extracytoplasmic moiety, RodZc. RodZn contains a predicted helix-turn-helix domain, whereas RodZc is thought to contain a coiled-coil region and a terminal C domain of unknown function. A single transmembrane domain separates RodZn from RodZc. Deletion of rodZ causes elongation of the nucleoid and leads to the production of polar minicells containing DNA. Here, we have studied the structure and function of RodZn and RodZc. We show that RodZn is a stable, folded, -helical domain. We discovered that the Y32A and L33A substitutions within the presumptive recognition helix (3) of the HTH motif, as well as the Y49A and F53A substitutions outside of the HTH motif (in 4) cause asymmetric cell division. However, only the substitutions in 4 cause sub-celular delocalization of RodZ. We suggest that 3 and 4 are used for a protein-protein or protein-DNA interaction important for cell division, whereas 4 is likely to contact a cytoskeletal component, presumably MreB. The polar cells formed by all the mutants are anucleate. We conclude that nucleoid elongation is not a prerequisite for asymmetric division. RodZc appears to be a largely unstructured domain, with some -sheet content, and is stabilized by the coiled-coil region. We show a homology relationship between RodZc and the NCX1 Na+/Ca2+ transporter and we found two residues within the C domain, G265 and N275, that are important for cell shape determination. These residues are predicted to be essential determinants of a claw-like motif, which may act as a binding site for an unknown ligand. Both the isolated RodZn and RodZc proteins are monomeric in solution. However, because full-length RodZ interacts with itself in a split-ubiquitin yeast two-hybrid assay, we suggest that it may dimerize or form higher order multimers in vivo.

Characterization of a Bacillus subtilis thermosensitive teichoic acid-deficient mutant: gene mnaA (yvyH) encodes the UDP-N-acetylglucosamine 2-epimerase.

The Bacillus subtilis thermosensitive mutant ts-21 bears two C-G-->T-A transitions in the mnaA gene. At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content. MnaA converts UDP-N-acetylglucosamine into UDP-N-acetylmannosamine, a precursor of the teichoic acid linkage unit.

Essential Bacillus subtilis genes.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

In Bacillus subtilis W23, the duet sigmaXsigmaM, two sigma factors of the extracytoplasmic function subfamily, are required for septum and wall synthesis under batch culture conditions.

The synthesis of poly(RboP), the main Bacillus subtilis W23 teichoic acid, is encoded by tarDF-tarABIJKL operons, the latter being controlled by two promoters designated PtarA-int and PtarA-ext. Analysis by lacZ fusions reveals that PtarA-int activity exhibits sharp increases at the beginning and end of the transition between exponential and stationary growth phase. As confirmed by mRNA quantification, these increases are mediated by ECF sigma factors sigmaX and sigmaM respectively. In liquid media, strain W23 sigX sigM double mutants experience serious difficulties in the transition and stationary growth phases. Inactivation of sigmaX- and sigmaM-controlled regulons, which precludes transcription from PtarA-int, leads to (i) delays in chromosome segregation and septation and (ii) a transient loss of up to 30% of the culture OD or lysis. However, specific inactivation of PtarA-int, leading mainly to a shortage of poly(RboP), does not affect growth while, nevertheless, interfering with normal septation, as revealed by electron microscopy. The different sigM transcription in strains W23 and 168 is discussed. In W23, expression of tarA and sigM, which is shown to control divIC, is inversely correlated with growth rate, suggesting that the sigM regulon is involved in the control of cell division.

The Bacillus subtilis Gne (GneA, GalE) protein can catalyse UDP-glucose as well as UDP-N-acetylglucosamine 4-epimerisation.

Mutations in the Bacillus subtilis gene that affect the activity of the uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 4-epimerase (EC 5.1.3.7) were shown to map to galE, the structural gene of the UDP-glucose (Glc) 4-epimerase (EC 5.1.3.2). This genetic evidence that the same enzyme can catalyse the epimerisation of hexoses as well as of their N-acetylated forms is confirmed by in vitro assays with purified enzyme. It appears that in B. subtilis, Gne (GneA, GalE) is involved in two distinct and essential functions, i.e., cell detoxification under certain growth conditions and the biosynthesis of anionic cell wall polymers. We discuss the evidence that such enzymes capable of utilizing both UDP-hexoses and UDP-N-acetylhexosamines are present in other organisms.

Bacillus subtilis alpha-phosphoglucomutase is required for normal cell morphology and biofilm formation.

Mutations designated gtaC and gtaE that affect alpha-phosphoglucomutase activity required for interconversion of glucose 6-phosphate and alpha-glucose 1-phosphate were mapped to the Bacillus subtilis pgcA (yhxB) gene. Backcrossing of the two mutations into the 168 reference strain was accompanied by impaired alpha-phosphoglucomutase activity in the soluble cell extract fraction, altered colony and cell morphology, and resistance to phages phi29 and rho11. Altered cell morphology, reversible by additional magnesium ions, may be correlated with a deficiency in the membrane glycolipid. The deficiency in biofilm formation in gtaC and gtaE mutants may be attributed to an inability to synthesize UDP-glucose, an important intermediate in a number of cell envelope biosynthetic processes.

Analysis of teichoic acid biosynthesis regulation reveals that the extracytoplasmic function sigma factor sigmaM is induced by phosphate depletion in Bacillus subtilis W23.

The expression of the Bacillus subtilis W23 tar genes specifying the biosynthesis of the major wall teichoic acid, the poly(ribitol phosphate), was studied under phosphate limitation using lacZ reporter fusions. Three different regulation patterns can be deduced from these beta-galactosidase activity data: (i) tarD and tarL gene expression is downregulated under phosphate starvation; (ii) tarA and, to a minor extent, tarB expression after an initial decrease unexpectedly increases; and (iii) tarO is not influenced by phosphate concentration. To dissect the tarA regulatory pattern, its two promoters were analysed under phosphate limitation: The P(tarA)-ext promoter is repressed under phosphate starvation by the PhoPR two-component system, whereas, under the same conditions, the P(tarA)-int promoter is upregulated by the action of an extracytoplasmic function (ECF) sigma factor, sigma(M). In contrast to strain 168, sigma(M) is activated in strain W23 in phosphate-depleted conditions, a phenomenon indirectly dependent on PhoPR, the two-component regulatory system responsible for the adaptation to phosphate starvation. These results provide further evidence for the role of sigma(M) in cell-wall stress response, and suggest that impairment of cell-wall structure is the signal activating this ECF sigma factor.

Novel Secretion Apparatus Maintains Spore Integrity and Developmental Gene Expression in Bacillus subtilis

Plos Genetics, 5(7): ARTe1000566

Bacillus subtilis bacteriophage SPP1 hexameric DNA helicase, G40P, interacts with forked DNA.

SPP1-encoded replicative DNA helicase gene 40 product (G40P) is an essential product for phage replication. Hexameric G40P, in the presence of AMP-PNP, preferentially binds unstructured single-stranded (ss)DNA in a sequence-independent manner. The efficiency of ssDNA binding, nucleotide hydrolysis and the unwinding activity of G40P are affected in a different manner by different nucleotide cofactors. Nuclease protection studies suggest that G40P protects the 5' tail of a forked molecule, and the duplex region at the junction against exonuclease attack. G40P does not protect the 3' tail of a forked molecule from exonuclease attack. By using electron microscopy we confirm that the ssDNA transverses the centre of the hexameric ring. Our results show that hexameric G40P DNA helicase encircles the 5' tail, interacts with the duplex DNA at the ss-double-stranded DNA junction and excludes the 3' tail of the forked DNA.

The Bacillus subtilis bacteriophage SPP1 G39P delivers and activates the G40P DNA helicase upon interacting with the G38P-bound replication origin.

Initiation of Bacillus subtilis bacteriophage SPP1 replication requires the phage-encoded genes 38, 39 and 40 products (G38P, G39P and G40P). G39P, which does not bind DNA, interacts with the replisome organiser, G38P, in the absence of ATP and with the ATP-activated hexameric replication fork helicase, G40P. G38P, which specifically interacts with the phage replication origin (oriL) DNA, does not seem to form a stable complex with G40P in solution. G39P when complexed with G40P-ATP inactivates the single-stranded DNA binding, ATPase and unwinding activities of G40P, and such effects are reversed by increasing amounts of G38P. Unwinding of a forked substrate by G40P-ATP is increased about tenfold by the addition of G38P and G39P to the reaction mixture. The specific protein-protein interactions between oriL-bound G38P and the G39P-G40P-ATPgammaS complex are necessary for helicase delivery to the SPP1 replication origin. Formation of G38P-G39P heterodimers releases G40P-ATPgammaS from the unstable oriL-G38P-G39P-G40P-ATPgammaS intermediate. G40P-ATPgammaS binds to the origin region, the uncomplexed G38P fraction remains bound to oriL, and the G38P-G39P heterodimer is lost from the complex. We demonstrate that G39P is a component of an oligomeric nucleoprotein complex which plays an important role in the initiation of SPP1 replication.

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7

No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) producedbyenterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.

Dosimetria esporular: Bacillus subtilis TKJ6312 como biossensor de radiação solar biologicamente ativa

Since 2000, spore dosimetry and spectral photometry have been performed in parallel at the Southern Space Observatory, São Martinho da Serra (Southern Brazil). A comparative study involving data from Punta Arenas - Chile (53.2º S), São Martinho da Serra (29.5º S), Padang - Indonesia (0.9ºS), Brussels - Belgium (50.9º N) and Kiyotake - Japan (31.9º N) from 2000 to 2006 is presented. The Spore Inactivation Doses presented the higher values in summer (973 ± 73 for Punta Arenas and 4,369 ± 202 for São Martinho da Serra, as well 1,402 ± 170 and 3,400 ± 1,674 for Brussels and Kiyotake, respectively). The simplicity, robustness and high resistance of bacterial spores makes the biosensor an potential biological tool for UV-B monitoring.

Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis

The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96% of the calcium, this strain thus has good potential for use on building materials.