Simone J. Séror

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 ≈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.

Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes.

PrkC was shown to be a eukaryotic‐like (Hanks‐type) protein kinase from Bacillus subtilis with a struc‐tural organization similar to that of the eukaryotic sensor Ser/Thr or Tyr kinases (e.g. the TGF β or PDGF receptors). The molecule consists of a catalytic domain located in the cytoplasm, joined by a single transmembrane‐spanning region (TMD) to a large extracellular domain. Using a genetic reporter system, involving the cI repressor of lambda, evidence was obtained indicating that PrkC forms a dimer, involving both the TMD and the external domain in dimerization. The purified catalytic domain of PrkC was shown to autophosphorylate and to phosphorylate an external target, MBP, in both cases on threonine. These two functions require the completely conserved K40 residue in subdomain II, which is essential for enzymatic activity. Importantly, both the mutant deleted for prkC and a K40R mutant exhibit decreased efficiency of sporulation and a significant reduction in biofilm formation, demonstrating that the catalytic activity of PrkC is necessary for these two developmental processes. In addition, we showed that the product of prpC, a PPM phosphatase encoded by the adjacent gene, co‐transcribed with prkC, is also required for normal biofilm and spore formation.

Comparative Analysis of the Development of Swarming Communities of Bacillus subtilis 168 and a Natural Wild Type: Critical Effects of Surfactin and the Composition of the Medium

The natural wild-type Bacillus subtilis strain 3610 swarms rapidly on the synthetic B medium in symmetrical concentric waves of branched dendritic patterns. In a comparison of the behavior of the laboratory strain 168 (trp) on different media with that of 3610, strain 168 (trp), which does not produce surfactin, displayed less swarming activity, both qualitatively (pattern formation) and in speed of colonization. On E and B media, 168 failed to swarm; however, with the latter, swarming was arrested at an early stage of development, with filamentous cells and rafts of cells (characteristic of dendrites of 3610) associated with bud-like structures surrounding the central inoculum. In contrast, strain 168 apparently swarmed efficiently on Luria-Bertani (LB) agar, colonizing the entire plate in 24 h. However, analysis of the intermediate stages of development of swarms on LB medium demonstrated that, in comparison with strain 3610, initiation of swarming of 168 (trp) was delayed and the greatly reduced rate of expansion of the swarm was uncoordinated, with some regions advancing faster than others. Moreover, while early stages of swarming in 3610 are accompanied by the formation of large numbers of dendrites whose rapid advance involves packs of cells at the tips, strain 168 advanced more slowly as a continuous front. When sfp+ was inserted into the chromosome of 168 (trp) to reestablish surfactin production, many features observed with 3610 on LB medium were now visible with 168. However, swarming of 168 (sfp+) still showed some reduced speed and a distinctive pattern compared to swarming of 3610. The results are discussed in terms of the possible role of surfactin in the swarming process and the different modes of swarming on LB medium.

Mass Spectrometry and Site-directed Mutagenesis Identify Several Autophosphorylated Residues Required for the Activity of PrkC, a Ser/Thr Kinase from Bacillus subtilis

We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity. Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive. Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity. In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism. When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region. These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism. The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways. This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family.

In situ localisation and quantification of surfactins in a Bacillus subtilis swarming community by imaging mass spectrometry

Surfactins are a family of heptacyclopeptides in which the C‐terminal carbonyl is linked with the β‐hydroxy group of a fatty acid acylating the N‐terminal function of a glutamic acid residue. The fatty acyl chain is 12–16 carbon atoms long. These compounds, which are secreted by the Gram‐positive bacterium Bacillus subtilis in stationary phase in liquid cultures, play an important role in swarming communities on the surface of agar media in the formation of dendritic patterns. TOF secondary ion MS (TOF‐SIMS) imaging was used to map surfactins within 16–17 h swarming patterns, with a 2 μm spatial resolution. Surfactins were mainly located in the central mother colony (the site of initial inoculation), in a ‘ring’ surrounding the pattern and along the edges of the dendrites. In the mother colony and the interior of the dendrites, surfactins with shorter chain lengths are present, whereas in the ring surrounding the swarm community and between dendrites, surfactins with longer fatty acyl chain lengths were found. A quantitative analysis by MALDI‐TOF MS showed a concentration gradient of surfactin from the mother colony to the periphery. The concentration of surfactin was ∼400 pmol/mL in the mother colony and ∼10 pmol/mL at the base of the dendrites, decreasing to 2 pmol/mL at their tips.

Characterization of PrpC from Bacillus subtilis, a Member of the PPM Phosphatase Family

We cloned the yloO gene and purified a His-tagged form of its product, the putative protein phosphatase YloO, which we now designate PrpC. This closely resembles the human protein phosphatase PP2C, a member of the PPM family, in sequence and predicted secondary structure. PrpC has phosphatase activity in vitro against a synthetic substrate, p-nitrophenol phosphate, and endogenous Bacillus subtilis proteins. The prkC and prpC genes are adjacent on the chromosome, and the phosphorylated form of PrkC is a substrate for PrpC. These findings suggest that PrkC and PrpC may function as a couple in vivo.

CpgA, EF-Tu and the stressosome protein YezB are substrates of the Ser/Thr kinase/phosphatase couple, PrkC/PrpC, in Bacillus subtilis.

The conserved prpC, prkC, cpgA locus in Bacillus subtilis encodes respectively a Ser/Thr phosphatase, the cognate sensor kinase (containing an external PASTA domain suggested to bind peptidoglycan precursors) and CpgA, a small ribosome-associated GTPase that we have shown previously is implicated in shape determination and peptidoglycan deposition. In this study, in a search for targets of PrkC and PrpC, we showed that, in vitro, CpgA itself is phosphorylated on serine and threonine, and another GTPase, the translation factor EF-Tu, is also phosphorylated by the kinase on the conserved T384 residue. Both substrates are dephosphorylated by PrpC in vitro. In addition, we identified YezB, a 10.3 kDa polypeptide, and a component of the stressosome, as a substrate for both enzymes in vitro and apparently in vivo. We propose that the PrpC/PrkC/CpgA system constitutes an important element of a regulatory network involved in the coordination of cell wall expansion and growth in B. subtilis.

Identification of genes required for different stages of dendritic swarming in Bacillus subtilis, with a novel role for phrC.

Highly branched dendritic swarming of B. subtilis on synthetic B-medium involves a developmental-like process that is absolutely dependent on flagella and surfactin secretion. In order to identify new swarming genes, we targeted the two-component ComPA signalling pathway and associated global regulators. In liquid cultures, the histidine kinase ComP, and the response regulator ComA, respond to secreted pheromones ComX and CSF (encoded by phrC) in order to control production of surfactin synthases and ComS (competence regulator). In this study, for what is believed to be the first time, we established that distinct early stages of dendritic swarming can be clearly defined, and that they are amenable to genetic analysis. In a mutational analysis producing several mutants with distinctive phenotypes, we were able to assign the genes sfp (activation of surfactin synthases), comA, abrB and codY (global regulators), hag (flagellin), mecA and yvzB (hag-like), and swrB (motility), to the different swarming stages. Surprisingly, mutations in genes comPX, comQ, comS, rapC and oppD, which are normally indispensable for import of CSF, had only modest effects, if any, on swarming and surfactin production. Therefore, during dendritic swarming, surfactin synthesis is apparently subject to novel regulation that is largely independent of the ComXP pathway; we discuss possible alternative mechanisms for driving srfABCD transcription. We showed that the phrC mutant, largely independent of any effect on surfactin production, was also, nevertheless, blocked early in swarming, forming stunted dendrites, with abnormal dendrite initiation morphology. In a mixed swarm co-inoculated with phrC sfp+ and phrC+ sfp (GFP), an apparently normal swarm was produced. In fact, while initiation of all dendrites was of the abnormal phrC type, these were predominantly populated by sfp cells, which migrated faster than the phrC cells. This and other results indicated a specific migration defect in the phrC mutant that could not be trans-complemented by CSF in a mixed swarm. CSF is the C-terminal pentapeptide of the surface-exposed PhrC pre-peptide and we propose that the residual PhrC 35 aa residue peptide anchored in the exterior of the cytoplasmic membrane has an apparently novel extracellular role in swarming.

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers.

The non-domesticated Bacillus subtilis strain 3610 displays, over a wide range of humidity, hyper-branched, dendritic, swarming-like migration on a minimal agar medium. At high (70 %) humidity, the laboratory strain 168 sfp+ (producing surfactin) behaves very similarly, although this strain carries a frameshift mutation in swrA, which another group has shown under their conditions (which include low humidity) is essential for swarming. We reconcile these different results by demonstrating that, while swrA is essential for dendritic migration at low humidity (30-40 %), it is dispensable at high humidity. Dendritic migration (flagella- and surfactin-dependent) of strains 168 sfp+ swrA and 3610 involves elongation of dendrites for several hours as a monolayer of cells in a thin fluid film. This enabled us to determine in situ the spatiotemporal pattern of expression of some key players in migration as dendrites develop, using gfp transcriptional fusions for hag (encoding flagellin), comA (regulation of surfactin synthesis) as well as eps (exopolysaccharide synthesis). Quantitative (single-cell) analysis of hag expression in situ revealed three spatially separated subpopulations or cell types: (i) networks of chains arising early in the mother colony (MC), expressing eps but not hag; (ii) largely immobile cells in dendrite stems expressing intermediate levels of hag; and (iii) a subpopulation of cells with several distinctive features, including very low comA expression but hyper-expression of hag (and flagella). These specialized cells emerge from the MC to spearhead the terminal 1 mm of dendrite tips as swirling and streaming packs, a major characteristic of swarming migration. We discuss a model for this swarming process, emphasizing the importance of population density and of the complementary roles of packs of swarmers driving dendrite extension, while non-mobile cells in the stems extend dendrites by multiplication.

The GTPase, CpgA(YloQ), a putative translation factor, is implicated in morphogenesis in Bacillus subtilis

YloQ, from Bacillus subtilis, was identified previously as an essential nucleotide-binding protein of unknown function. YloQ was successfully over-expressed in Escherichia coli in soluble form. The purified protein displayed a low GTPase activity similar to that of other small bacterial GTPases such as Bex/Era. Based on the demonstrated GTPase activity and the unusual order of the yloQ G motifs, we now designate this protein as CpgA (circularly permuted GTPase). An unexpected property of this low abundance GTPase was the demonstration, using gel filtration and ultracentrifugation analysis, that the protein formed stable dimers, dependent upon the concentration of YloQ(CpgA), but independent of GTP. In order to investigate function, cpgA was placed under the control of the pspac promotor in the B. subtilis chromosome. When grown in E or Spizizen medium in the absence of IPTG, the rate of growth was significantly reduced. A large proportion of the cells exhibited a markedly perturbed morphology, with the formation of swollen, bent or ‘curly’ shapes. To confirm that this was specifically due to depleted CpgA a plasmid-borne cpgA under pxyl control was introduced. This restored normal cell shape and growth rate, even in the absence of IPTG, provided xylose was present. The crystal structure of CpgA(YloQ) suggests a role as a translation initiation factor and we discuss the possibility that CpgA is involved in the translation of a subset of proteins, including some required for shape maintenance.