Characterization of Bacillus subtilis bacteriophages

Brodetsky, Anna M. (University of California, Los Angeles), and W. R. Romig. Characterization of Bacillus subtilis bacteriophages. J. Bacteriol. 90:1655-1663. 1965.-A group of six phages, SP5, SP6, SP7, SP8, SP9, and SP13, which use the Marburg strain of Bacillus subtilis as host was characterized. These phages, referred to as group 1, were examined for the following properties: host range, plaque morphology, stability, adsorption kinetics, one-step growth characteristics, calcium requirements, serum neutralization, thermal inactivation, and inactivation by ultraviolet irradiation. Five unrelated B. subtilis phages, SP3, SP10, PBS1, SP alpha, and SP beta, were included in the studies. When first isolated, none of the group 1 phages was able to replicate efficiently on B. subtilis SB19, a mutant of the "transforming" B. subtilis 168. Host range mutants capable of growth in SB19 were isolated for all of the group 1 phages except SP13, and are designated the "star" phages (SP5* through SP9*). For characterization, SB19 was used as host for the star phages, and another B. subtilis mutant, 168B, was host for SP13.     

Functional analysis of the Bacillus subtilis Zur regulon

The Bacillus subtilis zinc uptake repressor (Zur) regulates genes involved in zinc uptake. We have used DNA microarrays to identify genes that are derepressed in a zur mutant. In addition to members of the two previously identified Zur-regulated operons (yciC and ycdHI-yceA), we identified two other genes, yciA and yciB, as targets of Zur regulation. Electrophoretic mobility shift experiments demonstrated that all three operons are direct targets of Zur regulation. Zur binds to an approximately 28-bp operator upstream of the yciA gene, as judged by DNase I footprinting, and similar operator sites are found preceding each of the previously described target operons, yciC and ycdHI-yceA. Analysis of a yciA-lacZ fusion indicates that this operon is induced under zinc starvation conditions and derepressed in the zur mutant. Phenotypic analyses suggest that the YciA, YciB, and YciC proteins may function as part of the same Zn(II) transport pathway. Mutation of yciA or yciC, singly or in combination, had little effect on growth of the wild-type strain but significantly impaired the growth of the ycdH mutant under conditions of zinc limitation. Since the YciA, YciB, and YciC proteins are not obviously related to any known transporter family, they may define a new class of metal ion uptake system. Mutant strains lacking all three identified zinc uptake systems (yciABC, ycdHI-yceA, and zosA) are dependent on micromolar levels of added zinc for optimal growth.     

Characterization of Bacillus subtilis recombinational pathways

Recombination in Bacillus subtilis requires the products of numerous rec loci. To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele. On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups. Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mutations, while group beta comprises the addA and addB mutations. Group gamma consists of the recH and recP mutations. These results suggest that B. subtilis has multiple pathways for genetic recombination and that the products of the genes within the alpha, beta, and gamma epistatic groups are involved in these alternative recombination pathways. The RecA protein is required in all three pathways of intermolecular recombination.     

枯草芽胞杆菌L-天冬氨酸a脱羧酶的酶学性质

b-丙氨酸是一种重要的医药化工原料,目前主要依靠化学法进行生产。探寻更为环保和高效的生物生产法是未来研究的一个方向。L-天冬氨酸a脱羧酶 (PanD) 能特异地脱去L-天冬氨酸的a羧基,生成b-丙氨酸。本文比较了3种分别来源于大肠杆菌、谷氨酸棒状杆菌及枯草芽胞杆菌的PanD比酶活 (分别为0.98、7.52和8.4 U/mg)。后两者的最适pH均为6.5,最适反应温度分别为65 ℃及60 ℃。与目前研究最多的来源于大肠杆菌和谷氨酸棒状杆菌的PanD相比,来源于枯草芽胞杆菌的PanD具有更好的活性和热稳定性,具有更强的工业应用潜力。同时,本文对该酶特有的翻译后自剪切及机理性失活现象进行了分析和讨论。     

Evidence for Cyclic Di-GMP-Mediated Signaling in Bacillus subtilis

Cyclic di-GMP (c-di-GMP) is a second messenger that regulates diverse cellular processes in bacteria, including motility, biofilm formation, cell-cell signaling, and host colonization. Studies of c-di-GMP signaling have chiefly focused on Gram-negative bacteria. Here, we investigated c-di-GMP signaling in the Gram-positive bacterium Bacillus subtilis by constructing deletion mutations in genes predicted to be involved in the synthesis, breakdown, or response to the second messenger. We found that a putative c-di-GMP-degrading phosphodiesterase, YuxH, and a putative c-di-GMP receptor, YpfA, had strong influences on motility and that these effects depended on sequences similar to canonical EAL and RxxxR-D/NxSxxG motifs, respectively. Evidence indicates that YpfA inhibits motility by interacting with the flagellar motor protein MotA and that yuxH is under the negative control of the master regulator Spo0A～P. Based on these findings, we propose that YpfA inhibits motility in response to rising levels of c-di-GMP during entry into stationary phase due to the downregulation of yuxH by Spo0A～P. We also present evidence that YpfA has a mild influence on biofilm formation. In toto, our results demonstrate the existence of a functional c-di-GMP signaling system in B. subtilis that directly inhibits motility and directly or indirectly influences biofilm formation.     

Functional analysis of the sortase YhcS in Bacillus subtilis

Sortases of Gram-positive bacteria catalyze the covalent C-terminal anchoring of proteins to the cell wall. Bacillus subtilis, a well-known host organism for protein production, contains two putative sortases named YhcS and YwpE. The present studies were aimed at investigating the possible sortase function of these proteins in B. subtilis. Proteomics analyses revealed that sortase-mutant cells released elevated levels of the putative sortase substrate YfkN into the culture medium upon phosphate starvation. The results indicate that YfkN required sortase activity of YhcS for retention in the cell wall. To analyze sortase function in more detail, we focused attention on the potential sortase substrate YhcR, which is co-expressed with the sortase YhcS. Our results showed that the sortase recognition and cell-wall-anchoring motif of YhcR is functional when fused to the Bacillus pumilus chitinase ChiS, a readily detectable reporter protein that is normally secreted. The ChiS fusion protein is displayed at the cell wall surface when YhcS is co-expressed. In the absence of YhcS, or when no cell-wall-anchoring motif is fused to ChiS, the ChiS accumulates predominately in the culture medium. Taken together, these novel findings show that B. subtilis has a functional sortase for anchoring proteins to the cell wall.     

Characterization of recombination-deficient mutants of Bacillus subtilis

An isogenic set of "prophage-free," DNA repair-proficient and -deficient strains of Bacillus subtilis were characterized phenotypically. The mutant strains were provisionally classified into four categories on the basis of their sensitivity to DNA-damaging agents, their ability to release phage after lysogenization followed by damage to chromosomal DNA, and their impairment in genetic exchange. The properties of double Rec- mutants showed that recF and addA belong to different epistatic groups, whereas recF, recL, and recH fall into the same group. More than one pathway for genetic exchange might be operative in B. subtilis.     

Characterization of recF suppressors in Bacillus subtilis

A recF mutation renders Bacillus subtilis cells very sensitive to DNA-damaging agents. Such a recF defect is partially suppressed either by the presence of the recA73 mutation or by the presence of a plasmid-borne, heterologous, single-stranded DNA-binding (ssb) protein gene. Plasmids carrying ssb genes also suppressed the recR and recL defects. Our results suggest that suppression occurs by increasing recombinational repair. The effect of the suppressors may be at the level of induction of the SOS response.     

Characterization of the parB-Like yyaA Gene of Bacillus subtilis

We have characterized the yyaA gene of Bacillus subtilis, located near the origin of chromosome replication (oriC). Its protein product is similar to the Spo0J protein, which belongs to the ParB family of chromosome- and plasmid-partitioning proteins. Insertional inactivation of the yyaA gene had no apparent effect on chromosome organization and partitioning during vegetative growth or sporulation. Subcellular localization of YyaA by immunofluorescence microscopy indicated that it colocalizes with the nucleoid, and gel retardation studies confirmed that YyaA binds relatively nonspecifically to DNA. Overexpression of yyaA caused a sporulation defect characterized by the formation of multiple septa within the cell. This phenotype indicates that YyaA may have a regulatory role at the onset of sporulation.     

Molecular Characterization of the Flagellar Hook in Bacillus subtilis

The structure of the Gram-positive flagellum is poorly understood, and Bacillus subtilis encodes three proteins homologous to the flagellar hook protein from Salmonella enterica. Here we generated a modified B. subtilis hook protein that could be fluorescently stained using a cysteine-reactive dye. We used the fluorescently labeled hook to demonstrate that FlgE is the hook structural protein and that FliK regulated hook length. We further demonstrate that two proteins of unknown function, FlhO and FlhP, and the putative hook cap, FlgD, were required for hook assembly, such that when flhO, flhP, or flgD was mutated, hook protein was secreted into the supernatant. All mutants defective in hook completion resulted in homogeneously reduced σ(D)-dependent gene expression due to the action of the anti-sigma factor FlgM.     

Characterization of the Bacillus subtilis sporulation gene spoVK.

The sporulation gene spoVK of Bacillus subtilis was cloned by use of the insertional mutation spoVK::Tn917 omega HU8. The spoVK gene was shown to be the site of an incorrectly mapped mutation called spoVJ517. Thus, a separate spoVJ gene as defined by the 517 mutation does not exist and is instead identical with spoVK.     

Functional analysis of the Bacillus subtilis cysK and cysJI genes

The function of the Bacillus subtilis cysK and cysJI (previously designated yvgQR) genes, expected to be involved in the assimilatory sulfate reduction pathway, was investigated. A B. subtilis mutant with a deletion in the cysJI genes was unable to use sulfate or sulfite as sulfur source, which confirmed that these genes encode sulfite reductase. A mutant with a transposon insertion in the cysK gene, whose deduced protein sequence showed similarity to cysteine synthases, grew poorly on sulfate and butanesulfonate. A strain in which cysK and yrhA, a cysK paralog, were inactivated was unable to grow with sulfate. Whereas expression of the cysJI genes was induced by sulfate, expression of cysK was repressed both by sulfate and by cysteine.     

Genetic Characterization of Recombination-Deficient Mutants of Bacillus subtilis

Recombination-deficient (rec), radiation-sensitive mutations in Bacillus subtilis are grouped in at least seven distinct loci. Map positions are determined for six of these loci.