Biosurfactant production and surface translocation are regulated by PlcR in Bacillus cereus ATCC 14579 under low-nutrient conditions

Bacillus cereus ATCC 14579 can respond to nutrient changes by adopting different forms of surface translocation. The B. cereus ATCC 14579 DeltaplcR mutant, but not the wild type, formed dendritic (branched) patterns on EPS [a low-nutrient medium that contains 7.0 g K(2)HPO(4), 3.0 g KH(2)PO(4), 0.1 g MgSO(4).7H(2)O, 0.1 g (NH(4))(2)SO(4), 0.01 g CaCl(2), 0.001 g FeSO(4), 0.1 g NaCl, 1.0 g glucose, and 125 mg yeast extract per liter] containing 0.7% agar. The dendritic patterns formed by sliding translocation of nonflagellated cells are enhanced under low-nutrient conditions and require sufficient production of a biosurfactant, which appears to be repressed by PlcR. The wild-type and complemented strains failed to slide on the surface of EPS agar because of the production of low levels of biosurfactant. Precoating EPS agar surfaces with surfactin (a biosurfactant produced by Bacillus subtilis) or biosurfactant purified from the DeltaplcR mutant rescued the ability of the wild-type and complemented strains to slide. When grown on a nutrient-rich medium like Luria-Bertani agar, both the wild-type and DeltaplcR mutant strains produced flagella. The wild type was hyperflagellated and elongated and exhibited swarming behavior, while the DeltaplcR mutant was multiflagellated and the cells often formed long chains but did not swarm. Thin-layer chromatography and mass spectrometry analyses suggested that the biosurfactant purified from the DeltaplcR mutant was a lipopeptide and had a mass of 1,278.1722 (m/z). This biosurfactant has hemolytic activity and inhibited the growth of several gram-positive bacteria.     

Biofilm Formation by Bacillus cereus Is Influenced by PlcR, a Pleiotropic Regulator

The ΔplcR mutant of Bacillus cereus strain ATCC 14579 developed significantly more biofilm than the wild type and produced increased amounts of biosurfactant. Biosurfactant production is required for biofilm formation and may be directly or indirectly repressed by PlcR, a pleiotropic regulator. Coating polystyrene plates with surfactin, a biosurfactant from Bacillus subtilis, rescued the deficiency in biofilm formation by the wild type.     

The CasKR Two-Component System Is Required for the Growth of Mesophilic and Psychrotolerant Bacillus cereus Strains at Low Temperatures

The different strains of Bacillus cereus can grow at temperatures covering a very diverse range. Some B. cereus strains can grow in chilled food and consequently cause food poisoning. We have identified a new sensor/regulator mechanism involved in low-temperature B. cereus growth. Construction of a mutant of this two-component system enabled us to show that this system, called CasKR, is required for growth at the minimal temperature (T_min). CasKR was also involved in optimal cold growth above T_min and in cell survival below T_min. Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing the casKR genes in a ΔcasKR mutant restored its ability to grow at T_min. Although it was first identified in the ATCC 14579 model strain, this mechanism has been conserved in most strains of the B. cereus group. We show that the role of CasKR in cold growth is similar in other B. cereus sensu lato strains with different growth temperature ranges, including psychrotolerant strains.     

Differential Involvement of the Five RNA Helicases in Adaptation of Bacillus cereus ATCC 14579 to Low Growth Temperatures

Bacillus cereus ATCC 14579 possesses five RNA helicase-encoding genes overexpressed under cold growth conditions. Out of the five corresponding mutants, only the ΔcshA, ΔcshB, and ΔcshC strains were cold sensitive. Growth of the ΔcshA strain was also reduced at 30°C but not at 37°C. The cold phenotype was restored with the cshA gene for the ΔcshA strain and partially for the ΔcshB strain but not for the ΔcshC strain, suggesting different functions at low temperature.Bacillus cereus is a human pathogenic sporulated bacterium which is associated with emetic and diarrheal types of food-borne illnesses (4). B. cereus is widespread in the environment and in a wide range of foods. The growth domains of B. cereus strains range from psychrotrophic to nearly thermophilic and correlate with several phylogenetic clusters (15), which presumably permit B. cereus to colonize many different habitats with different thermal regimes. Many foods are stored refrigerated before consumption, and in such cases, B. cereus has to adapt to low-temperature conditions.B. cereus growth at low temperature takes place with a lag phase which may correspond to an adaptation phase (12). Cold is a stress which dramatically affects membrane fluidity, protein synthesis, and also the topology of nucleic acids (22). When exposed to low temperature, bacteria have to face a transient inhibition of protein synthesis mainly due to the presence of secondary structures in mRNA that are stabilized by cold conditions (16, 19). To overcome the translation interruption, cold-shocked cells synthesize cold-induced RNA helicases, which remove secondary structures from RNA duplexes in the presence of ATP, such as CsdA of Escherichia coli (19) or CshA of Bacillus subtilis (1). csdA and srmB deletion mutants of E. coli showed a cold-sensitive phenotype, and these RNA helicases have been described as involved in the biogenesis of the ribosomal 50S subunit at 20°C (10, 11). RNA helicases could also be involved in the degradation of mRNA by unwinding double-stranded mRNA, thereby allowing the action of RNase (8).We have recently shown that the deregulation of the expression of one RNA helicase gene of B. cereus ATCC 14579 increased the lag phase of B. cereus at a low temperature (7). In this context, our aim was to investigate the role of the five putative RNA helicases present in the genome of B. cereus ATCC 14579 in its adaptation at low temperature, close to the growth limit.     

Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A,spo0B,and spo0F in Phage AP50c Infection of Bacillus anthracis

In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.     

Exploring the Biosynthesis of Unsaturated Fatty Acids in Bacillus cereus ATCC 14579 and Functional Characterization of Novel Acyl-Lipid Desaturases

At low temperatures, Bacillus cereus synthesizes large amounts of unsaturated fatty acids (UFAs) with double bonds in positions Δ5 and Δ10, as well as Δ5,10 diunsaturated fatty acids. Through sequence homology searches, we identified two open reading frames (ORFs) encoding a putative Δ5 desaturase and a fatty acid acyl-lipid desaturase in the B. cereus ATCC 14579 genome, and these were named BC2983 and BC0400, respectively. Functional characterization of ORFs BC2983 and BC0400 by means of heterologous expression in Bacillus subtilis confirmed that they both encode acyl-lipid desaturases that use phospholipids as the substrates and have Δ5 and Δ10 desaturase activities. Thus, these ORFs were correspondingly named desA (Δ5 desaturase) and desB (Δ10 desaturase). We established that DesA utilizes ferredoxin and flavodoxins (Flds) as electron donors for the desaturation reaction, while DesB preferably employs Flds. In addition, increased amounts of UFAs were found when B. subtilis expressing B. cereus desaturases was subjected to a cold shock treatment, indicating that the activity or the expression of these enzymes is upregulated in response to a decrease in growth temperature. This represents the first work reporting the functional characterization of fatty acid desaturases from B. cereus.     

Deletion of the sigB Gene in Bacillus cereus ATCC 14579 Leads to Hydrogen Peroxide Hyperresistance

The sigB gene of Bacillus cereus ATCC 14579 encodes the alternative sigma factor σ^B. Deletion of sigB in B. cereus leads to hyperresistance to hydrogen peroxide. The expression of katA, which encodes one of the catalases of B. cereus, is upregulated in the sigB deletion mutant, and this may contribute to the hydrogen peroxide-resistant phenotype.     

Sporulation Temperature Reveals a Requirement for CotE in the Assembly of both the Coat and Exosporium Layers of Bacillus cereus Spores

The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H₂O₂ and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation.     

Mitochondrial Efficiency-Dependent Viability of Saccharomyces cerevisiae Mutants Carrying Individual Electron Transport Chain Component Deletions

Mitochondria play a crucial role in eukaryotic cells; the mitochondrial electron transport chain (ETC) generates adenosine triphosphate (ATP), which serves as an energy source for numerous critical cellular activities. However, the ETC also generates deleterious reactive oxygen species (ROS) as a natural byproduct of oxidative phosphorylation. ROS are considered the major cause of aging because they damage proteins, lipids, and DNA by oxidation. We analyzed the chronological life span, growth phenotype, mitochondrial membrane potential (MMP), and intracellular ATP and mitochondrial superoxide levels of 33 single ETC component-deleted strains during the chronological aging process. Among the ETC mutant strains, 14 (sdh1Δ, sdh2Δ, sdh4Δ, cor1Δ, cyt1Δ, qcr7Δ, qcr8Δ, rip1Δ, cox6Δ, cox7Δ, cox9Δ, atp4Δ, atp7Δ, and atp17Δ) showed a significantly shorter life span. The deleted genes encode important elements of the ETC components succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV), and some of the deletions lead to structural instability of the membrane-F₁F₀-ATP synthase due to mutations in the stator stalk (complex V). These short-lived strains generated higher superoxide levels and produced lower ATP levels without alteration of MMP. In summary, ETC mutations decreased the life span of yeast due to impaired mitochondrial efficiency.     

Innate immunity to yeast prions: Btn2p and Cur1p curing of the [URE3] prion is prevented by 60S ribosomal protein deficiency or ubiquitin/proteasome system overactivity

[URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ, and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p, or Btn3p are unchanged in rpl4aΔ, rpl21bΔ, or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild-type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ, or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems.     

Characterization of the <Emphasis Type="Italic">codY</Emphasis> gene and its influence on biofilm formation in <Emphasis Type="Italic">Bacillus </Emphasis><Emphasis Type="Italic">cereus</Emphasis>

The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.     

The Role of Glucosidase I (Cwh41p) in the Biosynthesis of Cell Wall β-1,6-Glucan Is Indirect

CWH41, a gene involved in the assembly of cell wall β-1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal α-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of β-1,6-glucan, we constructed a double mutant, alg5Δ (lacking dolichol-P-glucose synthase) cwh41Δ, and found that it has the same phenotype as the alg5Δ single mutant. It contains wild-type levels of cell wall β-1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41Δ single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the β-1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5Δ). The triple mutant alg5Δcwh41Δkre6Δ is viable, whereas the double mutant cwh41Δkre6Δ in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall β-1,6-glucan, characteristic of the cwh41Δkre1Δ double mutant, are not observed in the triple mutant alg5Δcwh41Δkre1Δ. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41Δ strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall β-1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway.     

Shared and independent roles for a Galpha(i) protein and adenylyl cyclase in regulating development and stress responses in Neurospora crassa

Growth and development are regulated using cyclic AMP (cAMP)-dependent and -independent pathways in Neurospora crassa. The cr-1 adenylyl cyclase mutant lacks detectable cAMP and exhibits numerous defects, including colonial growth habit, short aerial hyphae, premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. Evidence suggests that the heterotrimeric Gα protein GNA-1 is a direct positive regulator of adenylyl cyclase. Δgna-1 strains are female-sterile, and Δgna-1 strains have reduced apical extension rates on normal and hyperosmotic medium, greater resistance to oxidative and heat stress, and stunted aerial hyphae compared to the wild-type strain. In this study, a Δgna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. Δgna-1 cr-1 mutants have severely restricted colonial growth and do not produce aerial hyphae on plates or in standing liquid cultures. Addition of cAMP to plates or standing liquid cultures rescues cr-1, but not Δgna-1 cr-1, defects, which is consistent with previous results demonstrating that Δgna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the Δgna-1 mutation are sterile; however, unlike cr-1 and Δgna-1 strains, the Δgna-1 cr-1 mutant does not produce protoperithecia. The Δgna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < Δgna-1 < cr-1 = Δgna-1 cr-1, consistent with a cAMP-dependent process. Taken together, the results suggest that in general, GNA-1 and CR-1 regulate N. crassa growth and development using parallel pathways, while thermotolerance is largely dependent on cAMP.     

Functions of VPA1418 and VPA0305 Catalase Genes in Growth of Vibrio parahaemolyticus under Oxidative Stress

The marine foodborne enteropathogen Vibrio parahaemolyticus has four putative catalase genes. The functions of two katE-homologous genes, katE1 (VPA1418) and katE2 (VPA0305), in the growth of this bacterium were examined using gene deletion mutants with or without complementary genes. The growth of the mutant strains in static or shaken cultures in a rich medium at 37°C or at low temperatures (12 and 4°C), with or without competition from Escherichia coli, did not differ from that of the parent strain. When 175 μM extrinsic H₂O₂ was added to the culture medium, bacterial growth of the ΔkatE1 strain was delayed and growth of the ΔkatE1 ΔkatE2 and ΔkatE1 ΔahpC1 double mutant strains was completely inhibited at 37°C for 8 h. The sensitivity of the ΔkatE1 strain to the inhibition of growth by H₂O₂ was higher at low incubation temperatures (12 and 22°C) than at 37°C. The determined gene expression of these catalase and ahpC genes revealed that katE1 was highly expressed in the wild-type strain at 22°C under H₂O₂ stress, while the katE2 and ahpC genes may play an alternate or compensatory role in the ΔkatE1 strain. This study demonstrated that katE1 encodes the chief functional catalase for detoxifying extrinsic H₂O₂ during logarithmic growth and that the function of these genes was influenced by incubation temperature.     

Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation

Capnocytophaga ochracea is a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces. C. ochracea possesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) in Flavobacterium johnsoniae was shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins in F. johnsoniae have been identified in the genome of C. ochracea; therefore, the T9SS may be involved in biofilm formation by C. ochracea. Here we constructed three ortholog-deficient C. ochracea mutants lacking sprB (which encodes a gliding motility adhesin) or gldK or sprT (which encode T9SS proteins in F. johnsoniae). Gliding motility was lost in each mutant, suggesting that, in C. ochracea, the proteins encoded by sprB, gldK, and sprT are necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprB strains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-type C. ochracea were denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation of C. ochracea.     

Cardiolipin Is Dispensable for Oxidative Phosphorylation and Non-fermentative Growth of Alkaliphilic Bacillus pseudofirmus OF4

Cardiolipin (CL), a membrane phospholipid in bacteria and mitochondria, has been hypothesized to facilitate movement of protons on the outer surface of membranes in support of respiration-dependent ATP synthesis, oxidative phosphorylation (OXPHOS). If so, the high levels of membrane CL found in alkaliphilic bacteria, such as Bacillus pseudofirmus OF4, might facilitate its robust OXPHOS at pH 10.5, where the bulk protonmotive (PMF) force is low. To address the role of CL in Bacillus pseudofirmus OF4, we studied strains in which genes (cls) potentially encoding a CL synthase (CLs) were deleted: three single (ΔclsA, ΔclsB, and ΔclsC), one double (ΔclsA/B), and one triple (ΔclsA/B/C) mutant. Two-dimensional thin layer chromatography analyses of lipid extracts from ³²P-labeled strains showed that the wild-type CL content was 15% of total phospholipids at pH 10.5 versus 3% at pH 7.5 during log phase. The % CL was higher (28–33%) at both pH values during stationary phase. The clsA gene plays a major role in CL biosynthesis as no detectable CL was found in ΔclsA-containing mutants, whereas the CL precursor phosphatidylglycerol was elevated. The ΔclsB mutant exhibited no significant reduction in CL, but clsB expression was up-regulated and appeared to support growth at pH 7.5. In the absence of detectable CL, the alkaliphile showed no significant deficits in non-fermentative growth, respiration-dependent ATP synthesis, or salt tolerance. Minor deficits in respiration and ATP synthase assembly were noted in individual mutants. In long term survival experiments, significant growth defects were found in ΔclsA strains and the ΔclsC strain at pH 10.5.