Functional analysis of bc5181 in Bacillus cereus

UNIVERSITÀ DI PISA

Research Doctorate School in Biological and Molecular Sciences

DOCTORATE PROGRAM: BIOTECNOLOGY FOR HEALTH

Functional Analysis of bc5181 in Bacillus cereus

Candidato

Relatore

Dott.ssa Sokhna Aissatou Gueye Prof.ssa Emilia Ghelardi

Presidente del Programma di Dottorato

Prof. Mario Campa

Contents

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1.

Abstract 5

2.

Introduction 7

2.1 The Bacillus genus……….7

2.2 Bacillus cereus group………8

2.3 Virulence factors of B. cereus………..…..10

2.4 Bacterial motility………13

2.5 Stress response………14

2.5.1 Disinfectants………....15

2.5.1.a Peracetic acid………....16

2.6 YbjQ-like superfamily………...16

2.7 Aim of the thesis………....17

3. Materials and methods 19

3.1 Bacterial strains, plasmids and growth conditions………19

3.2 RNA isolation and RT-PCR analysis……….20

3.3 Strain construction……….21

3.4 Growth curves………23

3.5 Antimicrobial susceptibility………...23

3.6 Biochemical profiles………..24

3.7 Treatment with peracetic acid………....24

3.8 Motility and chemotaxis assays……….24

3.10 Detection of phosphatidylcholine-preferring phospholipase C (PC-PLC), proteases,

haemolysins, and L2 component of the haemolytic enterotoxin (HBL)……….. .26

3.11 Protein samples and gel electrophoresis………27

3.12 Biofilm formation………...27

3.13 Statistical analysis………..28

4. Results 29

4.1 bc5181: in silico analysis and gene expression………29

4.2 bc5181 does not affect growth, biochemical profile and antibiotic resistance…….30

4.3 Overexpression of bc5181 increases resistance to PAA treatment………34

4.4 bc5181 and motility………...31

4.5 Contribution of bc5181 to B. cereus pathogenicity and virulence……….37

4.6 Heterologous expression of bc5181 in B. subtilis………...40

5. Discussion 43

6. Conclusion 46

7. References 47

1. Abstract

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Bacillus cereus to disinfectant treatments showed a common up-regulation of stress-related

genes. In this study, we focused our attention on one of these genes (bc5181) that encodes a protein belonging to the YbjQ-like superfamily. Since bc5181 expression was shown to be induced by mild or lethal concentrations of peracetic-acid (PAA), we examined its role under PAA treatment by evaluating the phenotypic response of B. cereus strains lacking or overexpressing this gene. A bc5181 null mutant (MP23) and a strain carrying an additional IPTG-inducible copy of this gene (MP25) were constructed from B. cereus ATCC 14579 and comparatively assayed. Treatment of mid-exponential phase cells with two PAA concentrations revealed that the lack or overexpression of bc5181 have no significant effect on bacterial survival to the mild PAA concentration. However, when the lethal concentration was used, a high resistance to PAA was observed in the strain MP25 compared to the wild-type, more pronouncedly following IPTG induction. Unexpectedly, a lower increase in the resistance to the lethal PAA concentration was also observed in the bc5181 null mutant MP23 compared to the wild type.

To evaluate whether deprivation of bc5181 or increase in its expression could have other effects on B. cereus physiology, bacterial growth, carbohydrate utilization, antibiotic resistance, motility, chemotactic response, as well as virulence and pathogenicity of all strains were evaluated. Moreover, the effect of heterologous expression of bc5181 in B. subtilis, a microorganism naturally lacking a bc5181 homologue, was investigated.

While no significant effect of bc5181 on bacterial growth, biochemistry and antibiotic resistance was evidenced, bc5181 overexpression was shown to cause a defect in swimming motility, chemotactic response toward the chemo-attractant L-glutamine, and swarming motility. In addition, increase in bc5181 expression level caused a significant reduction in B.

cereus pathogenicity in an in vivo animal model of infection using the lepidoteran Galleria mellonella and in the production of some relevant B. cereus virulence factors, such as

phosphatidylcholine-preferring phospholipase C (PC-PLC), proteases and general haemolysins. Heterologous expression of bc5181 in B. subtilis was shown to increase the production of proteases and the propensity to develop biofilm communities by this organism.

In conclusion, bc5181 appears to be a non-essential stress-related gene whose overexpression increases resistance to PAA treatment, causes alterations in the motility and chemotactic behaviors, and leads to a reduction in B. cereus pathogenicity and virulence. The observation that heterologous bc5181 expression in a microorganism lacking ybjQ-like genes causes an increase in protease secretion and biofilm formation is also relevant, and further support a role of this gene in exerting a pleiotropic effect on bacterial cells.

2. Introduction

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2.1

The genus Bacillus

In 1835, Ehrenberg observed and described “Vibrio subtilis”. In 1872, Cohn renamed the organism Bacillus subtilis (Gordon, 1981). B. subtilis represented what was to become a large and diverse genus of bacteria named Bacillus. Bacteria belonging to this genus are Gram-positive bacilli, aerobes or facultative anaerobes, and usually motile for the presence of peritrichous flagella. They are also capable of forming heat resistant endospores, which are metabolically inert and can endure extreme conditions.

The genus Bacillus belongs to the family Bacillaceae (Priest, 1993) and it is unusually large. Hence, a further subdivision into six subgroups has been proposed (Priest, 1993). Since its discovery, the genus Bacillus has undergone considerable taxonomic changes. Starting off with two prominent and truly endospore-forming species, Bacillus anthracis and B. subtilis (until the early 1900s some taxonomists did not restrict the genus to endospore-forming bacteria), the number of species allocated to this genus increased to an incredible 146 in the 5th _{edition of Bergey’s Manual of Determinative Bacteriology (Bergey et al., 1939).} Meticulous comparative studies of Smith et al. (Smith et al., 1952) and Gordon et al. (Gordon

et al., 1973) on 1,114 strains of aerobic endospore-forming bacteria (AEFB) helped to reduce

this number to 22 well-defined species in the 8th _{edition of Bergey’s Manual of Determinative}

Bacteriology (Buchanan et al., 1974). A general new starting point for bacterial taxonomy

occurred in 1980 with the publication of the Approved Lists of Bacterial Names (Skerman et

al., 1980) where numerous taxonomic experts cooperated in establishing a comprehensive

and agreed upon list of accepted names of bacterial species.

The Bacillus genus includes a range of species with great interest for (1) industrial applications, such as production of biotechnological products (insect toxins, peptide antibiotics, enzymes for detergents, etc.) (Priest, 1993); (2) use of the spore as a model system

for studying bacterial cell differentiation and resistance to decontaminating agents or

treatments; and (3) the role of certain Bacillus species in causing human diseases.

Certain taxa of AEFB have received considerable taxonomic attention because of their outstanding applied importance. This is especially true for two groups of organisms: the B.

subtilis group and the B. cereus group.

2.2

Bacillus cereus group

The B. cereus group comprises the closely related species Bacillus cereus sensu stricto,

Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis (Jensen et al., 2003). Genotypic methods, such as full genomic

sequencing (Ivanova et al. 2003), amplified fragment length polymorphism (AFLP) (Ticknor et al., 2001), rep-PCR fingerprinting (Cherif et al., 2003), 16S rDNA and 23S rDNA analysis (Ash et al., 1991), multi-locus enzyme electrophoresis (MLEE) (Ash and Collins

1992; Helgason et al., 2000), multi-locus sequence typing (MLST) (Helgason et al., 2004;

Priest et al., 2004; Tourasse et al.,2006) and suppression subtractive hybridization (SSH) (Radnedge et al., 2003), have been extensively used to study phylogenetic relationships among bacteria of the B. cereus group. The genetic relatedness between members of the group is so close that from a strictly phylogenetic point of view they can be considered as a single species. Hence, species differentiation within the B. cereus group is particularly complex, and a polyphasic approach is required for clear identification. Central to the identification of the various members of the B. cereus group is the analysis of phenotypic characteristics and pathogenicity traits, as well as of the presence of extra-chromosomal elements, which reflect the species’ virulence spectra.

B. cereus sensu stricto is a common soil bacterium that shows the ability to undergo a switch process from a single-cell to a multicellular phenotype, which allows it to translocate through the soil (Vilain et al., 2006). This morphogenetic phase is analogous to the swarming behaviour on agar media (Senesi et al., 2002). B. cereus can behave as a symbiont in colonizing invertebrate guts, an environment that has been suggested to be its natural habitat

(Margulis et al., 1998; Jensen et al., 2003). As a pathogen, B. cereus produces a great variety of toxins and enzymes that possess haemolytic, cytotoxic, dermonecrotic, and vascular permeability activities, as demonstrated by in vitro and in vivo assays (reviewed in Bottone

et al., 2010). These virulence factors make B. cereus able to cause mild gastrointestinal to

severe systemic infections such as endophthalmitis, meningitis or septicaemia, which are particularly harmful for immunocompromised patients, newborns, and patients with surgical wounds (Kotiranta et al., 2000). The ability of B. cereus to produce infections in both human and non-human species is attributed to a wide array of mechanisms, including adherence (Kotiranta et al., 1998), invasion, evasion of host defences and damage to host cells(Beecher

and Wong 1997; Hardy et al., 2001; Haug et al., 2010).

B. thuringiensis is also frequently isolated from the soil. B. thuringiensis is an entomopathogenic bacterium, and it is the most commonly used commercial biopesticide worldwide (Soberon et al., 2007). Despite this common use, it can behave as a pathogen,

being able to cause wound, lung, and eye infections (Samples and Buettner, 1983; Hernandez

et al., 1998; Ghelardi et al., 2007). Its identification and classification are based on the

production of insecticidal proteinaceous toxin crystals (Cry and Cyt proteins) during sporulation (Schnepf et al., 1998; Aronson, 2002). The Cry and Cyt toxins are different and exhibit variable specificities towards the larvae of different classes of insects (reviewed in

Whiteley and Schnepf, 1986; Schnepf et al., 1998).

B. anthracis is a highly monomorphic species within the B. cereus group, showing very little genetic variation (Keim et al., 1997; Van Ert et al., 2007). In the environment, the bacterium primarily exists as a highly stable, dormant spore in the soil. Nevertheless, it has been claimed that the organism can grow and persist outside the host, in the rhizosphere of plants (Saile and Koehler, 2006). The majority of B. antracis strains can be easily distinguished from other characterized strains of the B. cereus group by the absence of haemolytic activity, when grown on blood agar, and penicillin sensitivity (Villas Bôas et al., 2007). B. anthracis is the cause of anthrax that primarily is a disease of herbivores, but may also cause isolated cases of infections in humans. Human anthrax takes three forms: cutaneous, gastro-intestinal, or inhalational (reviewed in Mock and Fouet, 2001). While the cutaneous form is easily treatable

with antimicrobials, the gastrointestinal and inhalational forms of the disease are more severe, as shown by the mortality rates following ingestion of meat from animals died from anthrax disease (reviewed by Beatty et al., 2003), as well as in a bioterroristic attack (Jernigan et al., 2001). Anthrax disease is primarily caused by two virulence factors: (1) a three components toxin constituted by the protective antigen (PA), the lethal factor (LF), and the edema factor

(EF) (Mock and Fouet, 2001; Mock and Mignot, 2003; Young and Collier, 2007), and (2) the

presence of a poly-γ-D-glutamic acid (polyglutamate) capsule which is important for B.

anthracis survival in the host, as it helps the bacterium to evade the host immune system by

protecting vegetative cells from phagocytotic killing during infection (Preisz, 1909; Drysdale

et al., 2005; Candela and Fouet, 2006; Richter et al., 2009)

B. weihenstephanensis can be differentiated from B. cereus by its psychrotolerant growth as well as characteristic sequence signatures in rDNA genes and in the gene encoding the cold shock protein cspA (Lechner et al., 1998). Pathogenicity of B. weihenstephanensis strains is generally regarded as low (Stenfors et al., 2002), but strains producing the cereulide toxin characteristic of emetic B. cereus strains, have been described (Thorsen et al., 2006).

B. mycoides (Flugge, 1886; Lewis, 1932) and B. pseudomycoides (Nakamura, 1998) are non-motile bacteria characterized by their ability to grow as filaments (rhizoidal phenotype) due to cells that remain in contact after division at the level of cell wall septa. B. mycoides can promote plant health by stimulating plant-host or mutualistic symbiosis (Choudahry and Johri, 2009). Nevertheless, B. mycoides is capable of producing virulence factors, such as hemolysin III, similar to those produced by B. cereus (Baida et al., 1997).

2.3 Virulence factors of B. cereus

B. cereus can cause food poisoning and various opportunistic and nosocomial infections. The

pathogenicity of B. cereus is intimately associated with tissue-destructive/reactive exoenzyme production. B. cereus can cause two types of food poisoning, one resulting in vomiting through the action of the emetic toxin cereulide and the other resulting in diarrhoea

Arnesen et al., 2008). Cereulide is a toxic peptide that, if present in the food at the time of ingestion, may cause vomit (Agata et al., 2002). The emetic toxin-encoding gene is located on a plasmid, pCER270 (Hoton et al., 2005; Ehling-Schulz et al., 2006). The presence of live bacterial cells is not required for the occurrence of the emetic syndrome. Regarding the diarrhoeic syndrome, it is unclear if the enterotoxins are present in food or are produced in the small intestine by the live bacteria. However, enterotoxins are unstable at pH less than 4 and can be degraded by pepsin, trypsin and chymotrypsin (Granum, 1994). So it is most likely that they are produced in the small intestine. Five toxins have been proposed as potential causes for the diarrhoeic syndrome: hemolysin BL (HBL, three-component toxin), nonhemolytic enterotoxin (NHE, three-component toxin), enterotoxin T (BceT), enterotoxin FM (EntFM) and cytotoxin K (CytK, single-component toxin of the β-barrel pore-forming toxin family), but only three (HBL, NHE and CytK) have been related to foodborne outbreaks (Agata et al.1995; Lund et al. 2000; Lund and Granum 1997; Stenfors Arnesen et al., 2008; Schoeni and Lee Wong, 2005) and are encoded by genes carried on the chromosome.

Other toxins produced by B. cereus include hemolysins (hemolysin II [HlyII] and III [HlyIII]) and phospholipase C (PLC). Three variants of PLC have been characterized: phosphotidylinositol hydrolase (PIH or PI-PLC), phosphotidylcholine hydrolase (PC-PLC) and sphingomyelinase (SMase) (Ivanova et al., 2003). Phospholipase C and hemolysins produced by B. cereus are necrotizing toxins that mimic the effects of some staphylococcal or clostridial toxins, resulting in invasive, non-gastrointestinal infections (Turnbull and Kramer 1983).

B. cereus may also produce other enzymes that may be relevant for virulence, which

include proteases, notably metalloproteases (Cadot et al., 2010; Guillemet et al., 2010), and other degradative enzymes. These enzymes are supposed to play a role in the establishment and development of infection, and in circumventing the host immune system.

Among additional virulence factors, the transcription factor Phospholipase C Regulator (PlcR) is involved in the expression of most known virulence factors in B. cereus, including HBL, Nhe,CytK, PLCs (Gohar et al. 2008) (Fig. 1). Most likely, the toxins can act synergistically to cause B. cereus food-borne disease (Stenfors Arnesen et al., 2008).

Figure 1. Schematic representation of the transcriptional regulator PlcR

2.3.1 Galleria mellonella: an

in vivo model for assessing bacterial

pathogenesis

Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial pathogens including bacteria, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens, Pseudomonas aeruginosa,

Listeria monocytogenes, Enterococcus faecalis, B. cereus or B. thuringiensis and fungi

(Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) (Jander et al., 2000; Purves

et al., 2010; Chadwick, 1967, Chadwick et al., 1990 and Ramarao et al., 2012). The use of an

insect model has a number of advantages over traditional mammalian models of infection. As an invertebrate, G. mellonella is not subjected the ethical limitations. In addition, the larvae can be easily maintained, infected by injection without anaesthesia, can undergo pre-treatment with chemical inhibitors (Banville et al., 2012) and sustain incubation at 37 °C (Mowlds et al., 2008). In particular, larvae of the great wax moth G. mellonella are widely used for the assessment of bacterial pathogenicity in vivo, since the invertebrate immune system functionally resembles the mammalian innate immune response to bacterial infections (Lemaitre and Hoffman, 2007). In previous work with B. cereus using insect infection models, the results were highly comparable to those obtained in a mouse model (Agaisse et al., 1999) (Fig.2).

A B

Figure 2. Alive (A, white) and dead (B, black) G. mellonella larvae.

2.4 Bacterial motility

In a world of limited resources, motility is an advantage, as it allows the organism to migrate towards nutrients and other favourable conditions, and away from unfavourable ones. Bacterial translocation can occur in a variety of ways: swimming, swarming, gliding, twitching, sliding and darting, depending on the organism and the properties of the surface or surrounding medium (Henrichsen, 1972; Jarrell and McBride, 2008). Many bacteria have flagella, which enable them to move by swimming in liquid media or swarming on a solid moist surface. However, the production and maintenance of flagella is structurally complicated and requires energy and amino acid resources (McCarter, 2006). Therefore, flagellar activity is under strict regulatory control (Smith and Hoover, 2009).

During flagella assembly, flagellin monomers, that constitute the building blocks of the filament structure, are transported through the hollow fibre and added to its growing distal end. The coordinate rotational direction of flagella governs the directional movement of the bacterium and is controlled by a phosphorelay signalling cascade receiving input from several two-component systems sensing environmental factors such as chemical concentration, oxygen levels, pH, the intensity or wavelength of light, temperature, and the presence of nutrients and integrate the signals to move towards or maintain the cells in optimum conditions for growth and division. Cells react to spatial gradients of chemo-attractants by increasing the frequency of tumbling in response to decreasing gradients and decreasing the frequency of tumbling in response to increasing gradients. Chemical gradients are sensed by a family of methyl-accepting chemotaxis receptors

.

As mentioned previously, flagellar biosynthesis consumes energy and amino acid resources and it is usually tightly controlled. The composition of the regulatory networks varies between species, as reviewed by Smith and Hoover (2009) and McCarter (2006).

B. cereus, B. thuringiensis, and B. weihenstephanensis are known to exhibit flagellar

motility, but its regulation at the molecular level is not well known. As shown previously, regulation is likely to be different from what is observed in B. subtilis, but may be partly similar to that of Listeria monocytogenes. Genes involved in chemotaxis are also reported to be organized differently in B. subtilis and B. cereus (Celandroni et al., 2000).

For many pathogenic bacteria, flagellum-dependent motility and chemotaxis are crucial factors in the process of colonization of the host organism and establishment of a successful infection (reviewed in references Moens et al., 1996 and Ottemann and Miller 1997).

2.5 Stress response

In natural environments, bacteria are continuously exposed to different types of stress, and they are equipped to handle temperature fluctuations, various types of chemical stresses (oxidative stress, high and low pH values, high salt concentrations, exposure to ethanol and other chemicals), and desiccation, as well as attacks from hostile microorganisms and host

immune systems (Abee and Wouters, 1999; Hecker et al., 2007). To increase their chance to

survive, bacteria evolved various mechanisms enabling them to face stresses and to maintain proper cellular physiology. In general, they undergo deep changes by a concerted reprogramming of the expression of several genes, which leads to the synthesis of proteins that are utilized for a rapid and optimal adjustment to a particular stress.

One response to different stress conditions is the induction of general stress proteins (GSPs) (Hecker, et al., 1996; Hecker and Völker, 1990; Völker et al., 1994). It is evident that the majority of the general stress genes belong to the large σB_{-dependent genes.}

The genes involved in different stress responses vary. Some genes are specific to one particular stress response, while other are activated by several types of stresses (Periago et al., 2002). In Gram-positive microorganisms, stress responses have been extensively studied in

group, particularly B. cereus ATCC 14579 (Browne and Dowds, 2001; den Besten et al., 2009; den Besten et al., 2006; Mols et al., 2007; Periago et al., 2002; van Schaik et al., 2004; van Schaik et al., 2007), revealing extensive adaption to severe stress by pre-exposure to mild stress and also that adaption leads to a large degree of cross-protection between various types of stress.

Transcription of many stress response genes is induced by heat. Such genes are considered part of the heat shock response, though their functions may be diverse. In B.

subtilis, the heat-inducible genes are classified according to their regulatory mechanism

(Derre et al., 1999; Hecker et al., 1996). This classification has been adopted also by related species.

2.5.1 Disinfectants

Besides antibiotics, other agents are often used to control bacterial growth. Those used to control bacteria on inanimate objects are called disinfectants. Those used on living tissue, such as skin, are called antiseptics. Antiseptics and disinfectants (biocides) are widely employed in controlling hospital infection. Their activity depends upon several factors, notably concentration, period of contact, pH, temperature, the type, nature and numbers of microorganisms to be inactivated and the presence of organic soil or other interfering material.

Antibacterial products can act on microorganisms in two different ways: inhibiting their growth (bacteriostatic, fungistatic) or exerting a lethal action (bactericidal or fungicidal effects). Only the lethal effects are of interest in disinfection.

Bacteria vary considerably in their response to antiseptics and disinfectants. Resistance is often intrinsic in nature, and it is found in bacterial spores, mycobacteria and Gram-negative bacteria. Resistance may also be acquired, either by mutation or by the acquisition of genetic elements. However, disinfectants are usually complex formulations of active molecules, sometimes also containing co-solvents, chelating agents, acidic or alkaline agents, or surface-active or anti-corrosive products.

2.5.1.a Peracetic acid

PAA (Fig.3) is considered a potent biocide, more active than hydrogen peroxide (H2O2), being

sporicidal, bactericidal, virucidal, and fungicidal at low concentrations (<0.3%) (Block, 1991).

Figure 3. Peracetic acid molecule

PAA also decomposes to safe by-products (acetic acid and oxygen) but has the added advantages of being free from decomposition by peroxidases, unlike H2O2, and remaining

active in the presence of organic loads (Lensing and Oei, 1984; Malchesky 1993). Its main application is as a low-temperature liquid sterilant for medical devices, flexible scopes, and hemodialyzers, but it is also used as an environmental surface sterilant (Crow 1992; Malchesky 1993). Similarly to H2O2, PAA probably denatures proteins and enzymes and

increases cell wall permeability by disrupting sulfhydryl (-SH) and sulfur (S-S) bonds (Baldry and Fraser, 1988; Block, 1991).

2.6 YbjQ-like superfamily

The YbjQ-like superfamily constitutes a large family of conserved proteins, shared by several prokaryotes and eukaryotes. Conformationally, the YbjQ-like superfamily belongs to the class of alpha and beta proteins (a+b), mainly constituted by antiparallel beta sheets folding in a dodecin subunit-like manner (Fig. 4). The dodecin monomers adopt a simple βαββ-fold and assemble to hollow sphere-like dodecameric complexes. The dodecin subunit-like fold has the following 6 subcategories: 1) amyloid beta a4 protein copper binding domain, 2) D-lysine 5,6-aminomutase beta subunit KamE, N-terminal domain, 3) dodecin-like, 4) YdgH-like, 5) N-terminal, heterodimerisation domain of RBP7 (RpoE), and 6) YbjQ-like.

Although these proteins are widely diffused, no functional information is available so far for the YbjQ-like family. It has been suggested that dodecin-like proteins may contribute to the protection against reactive oxygenic species by the sequestration of free cytosolic iron species (Reindel et al., 2002). Taking into consideration the structure and the wide diffusion among pathogenic and soil bacteria, a function for the dodecins in flavin storage or protection against radical or oxygenic stress has been suggested (Boris Bieger et al., 2003).

Recently, conformational studies of this superfamily of proteins report that YbjQ-like proteins constitutes homopentameric structures, which behave as metamorphic proteins (Andreeva and Murzin 2010). Indeed, they may adopt different conformations for the same amino-acid sequence under native conditions (Murzin, 2008).

Similarly to many pathogenic microorganisms, including negative and Gram-positive bacteria, fungi, and protists, B. cereus possess a like gene, named bc5181. ybjQ-like genes are widely distributed in the Bacillus genus and are present in all the species of the “Bacillus cereus group” and in some microorganisms belonging to the B. subtilis group such as B. licheniformis and B. amyloliquefaciens, B. subtilis excluded.

2.7 Aim of the thesis

• Analyze the contribution of bc5181 in the resistance of B. cereus ATCC 14579 to peracetic acid treatment;

• Evaluate whether bc5181 could have a role in B. cereus physiology, influencing phenotypic traits, motility, chemotactic response, pathogenicity and virulence of this organism;

• Study the effect of bc5181 in a microorganism that does not contain ybjQ-like genes, e.g.

S.flexneri ---MQFSTTPTLEGQTIVEYCGVVTGEAILGANIFRDFFAGIRDIVGGRSGAYEKELRKAREIAFEELGSQARALGADAVVGIDIDYETVGQNGSMLMVSVSGTAVKTRR-- 107

V.cholerae ---MIVTTTPNIEGKRIVRYCGVIAGEAILGANIFKDLFAGIRDIVGGRSGTYERELEKARAIALEELQQHAVALGANAVVGIDLDYETFGKANGMLMVSVSGTAVVVE--- 106

P.putida ---MIISTTSQLEGRPIAEYLGVVSSESVQGINFVRDFFTRFRDFFGGRSQTLESALREAREQATEELKARARQLQADAVVGVDFEISMPSVQGGMVVVFATGTAVRLK--- 106

F.tularensis --MKDILLTTTENIPGYRIVKVLGIVSANTVRARHIGKDFVASLRNIVGGEIKEYAELLQHSRRYVLDKLKEEARKLGANAVVGLRFSTASIMQRAAEILAYGTAVVVEES--- 109

T.thermophilus MGVMEILVSTLEAVPGYRVAQVLGVVKGSTVRSKHLGKDLLAGLRTLVGGELPEYTEMLQEAREVAEARMLEEARRLGAHAVLGVRYATASVMQGAAEILVYGTAVRLEPARER 114

C.tetani ---MILSNLSYIDNK-ELETINIVEGVCVFSKNVGKDIMASFKNVVGGEIKSYSEMVRDVKDTAVKKMVEEAKNLGADAVINIRYAMTSMSQ-GSTLAVIVSGTAVKVKGE- 106 B.cereus ---MIVTTTSTIQGKEIIDYVDIVNGEAIMGANIVRDLFASVRDVVGGRSGAYESKLKEARDIAMEEMKTLARQKNANAIVGIDVDYEVVR--EGMLMVAVSGTAVRI---- 103

: .:. : . .:: . : . :. :*:.: .: ..**. :.. : . .: * *.*::.: . : . :.. :

Figure 4. Overall Structure of the B. cereus YbjQ-like protein

(A) Alignment of the amino acid sequences of YbjQ-like orthologs. The alignment was performed by CLUSTALW and includes the YbjQ sequences found in the genomes of Bacillus cereus ATCC 14579 (BC5181),

Shigella flexneri (SFV_0854), Vibrio cholerae (VC0395_0288), Pseudomonas putida (PPS_2539), Francisella

tularensis (FTW_0852), Thermus thermophilus(TTC1581), and Clostridium tetani (CTC01500). The secondary structure of the B. cereus YbjQ-like is drawn beneath the alignment. Blue, pink, red, and green amino acid symbols indicate acidic, basic, apolar, and polar residues, respectively.

(B) Secondary structure of the YbjQ-like protein fold (C) Tertiary structure of the YbjQ-like protein fold

(D) Metamorphic proteins. (a, b) Side-by-side comparaison of alternatively folded subunits of the DUF74 pentamer.

Key:

Sec. struc: Helices labelled H1, H2, ... and strands by their sheets A, B, ...Helix Strand Motifs: beta turn beta hair

B

C

D A

3. Materials and methods

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3.1 Bacterial strains, plasmids and growth conditions

The B. cereus completely sequenced strain ATCC 14579 obtained from the American Type Culture Collection (ATCC) was used. A bc5181 null mutant (MP23) and a strain carrying an additional copy of this gene under the control of an IPTG-inducible promoter (MP25) were constructed from B. cereus ATCC 14579. B. subtilis 168 was used to construct a B. subtilis strain containing a copy of bc5181 under the control of the Pspac trascritional control (MP26). All the strains constructed for this study are reported in Table 1. Bacteria were grown in tryptone-NaCl (TrB; 1% tryptone, 0.5 % NaCl), brain heart infusion (BHI), or the same media supplemented with 0.1% glucose (TrBG, BHIG), or Luria-Bertani broth (LB). Media were routinely solidified with 1 % bacteriological agar, unless otherwise specified. For selection of

B. cereus transformants, appropriate antibiotics (25 μg erythromycin ml−1 _{or 30 o 200 μg} kanamycin ml−1_{) were added to the media. The E. coli strain TOP10 (Invitrogen) was used} for general cloning strategies, while strain SCS110 (Stratagene) was used as an intermediary host in B. cereus transformation experiments. E. coli strains were grown at 37 °C in BHI supplemented with ampicillin (50 μg ml−1_{) when required.}

Table 1. Bacterial strains and plasmids used in this study

3.2 RNA isolation and RT-PCR analysis

Total RNA was extracted from B. cereus cells grown in TrB. Cell suspensions were added to an equal volume of ice-cold methanol and incubated at room temperature for 5 min. Bacteria were collected by centrifugation at 4,000 × g at 4°C for 5 min, and the cell pellets

Strain or plasmid Genotype or characteristic Source or reference

B. cereus

ATCC 14579 Wild-type strain ATCC

MP23 ATCC 14579 bc5181::aphA3 Km This study

MP25 Derivative of ATCC 14579, containing pDGbc5181 This study

MP28 Derivative of ATCC 14579, containing pDG148 This study

B. subtilis

168 Wild-type strain without bc5181 Spizizen (1958)

MP26 Derivative of 168, containing pDGbc5181 This study

E. coli

SCS110 rpsL (Strr_{) thr leu endA thi-1 lacY galK galT ara tonA tsx dam dcm} supE44 Δ(lac-proAB) [F′ traD36 proAB lacIq_{Z ΔM15]}

Stratagene

TOP10 F− _{mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR}

recA1 endA1 araΔ139 Δ(ara, leu)7697 galU galK λ− _rpsL(Strr₎ nupG

Invitrogen

Plasmids

pMAD AmpR_{, Em}R _{shuttle vector, thermosensitive origin of replication Arnaud et al. (2004)}

pDG148 Expression vector with IPTG-inducible spac promoter (Pspac) KmR

AmpR_;

Stragier et al. (1988)

pDG783 aphA3 gene 1.5-kb kanamycin resistance cassette in pSB118; AmpR_,

KmR

Guèrout-Fleury et al.

(1995)

pEFAKB1 2.9-kb BamHI-NcoI fragment containing the 801-bp bc5181

upstream region, aphA3 Km, and the 638-bp bc5181 downstream region, inserted into pMAD/BamHI-NcoI

This study

(~109 _{bacterial cells) were rapidly frozen on dry ice and stored at −80°C until use. RNA}

extraction (performed with the RNeasy Midi kit; Qiagen, Hilden, Germany), RNA

quantification, and cDNA synthesis and labelling were performed as described previously (Ghelardi et al., 2002). The concentration, integrity and purity of RNA samples were determined spectrophotometrically and by visual inspection of agarose gels. Purified RNA (100 ng) was used as a template in one-step RT-PCR with the cMaster RTplus_{PCR system} (Eppendorf), according to the manufacturer's instructions. The primers used in different reactions, listed in Table 2 were: BC5181F2/BC5181R1, for the amplification of bc5181 cDNA and ywjAF2/BC5181R1, for the amplification of a cDNA fragment containing both

bc5181 and ywjA; The RT reactions were carried out at 42°C for 60 min in a thermal cycler,

immediately followed by 30 cycles of PCR, consisting of 30 s at 94°C, 30 s at 52°C and 1 min 30 s at 72°C, and ending with a 10 min incubation at 72°C. To exclude DNA contamination in RNA samples, reactions in which RT enzyme was replaced by RNase-free water (negative controls) were performed. As positive control, genomic DNA was used as template. The RT-PCR products were analyzed by agarose gel electrophoresis.

3.3 Strain construction

The thermosensitive pMAD vector (Arnaud et al., 2004) was used to construct the recombinant plasmid used for bc5181 deletion by allelic exchange in B. cereus ATCC 14579 (type strain, GenBank accession number AE016877). A kanamycin resistance (Kmr_{) cassette} (aphA3 gene) and regions upstream (portion of ywjA) and downstream (portion of insK) of bc5181, both overlapping a portion of bc5181 in B. cereus ATCC 14579, were used.

A 638–bp PstI-NcoI fragment (extending from positions + 89 to - 537 with respect to the

bc5181 translation initiation site) termed fragment A and an 801bp BamHI-HindIII fragment

(extending from positions -101 to + 688 with respect to the bc5181 stop codon) termed fragment B were generated by PCR, using the primer pairs BC5181L1P/ywjAR1N and insKU2B/ BC5181R1H respectively (Table 2). The aphA3 kanamycin resistance gene (Kmr₎ was extracted from pDG783 by enzymatic restriction with PstI and HindIII enzymes (Guerout-Fleury et al., 1998). Fragments A and B, as well as the aphA3 gene were simultaneously ligated in pMAD, previously digested with BamHI and NcoI enzymes. The

resulting vector, pMAD-fragmentA-aphA3Km-fragmentB termed pEFAKB1 was inserted into E. coli TOP 10, on each side of the 1.5-kb PstI-HindIII fragment carrying the aphA3 gene, between the sites of the thermosensitive plasmid pMAD, conferring erythromycin resistance to B. cereus and ampicillin resistance to E. coli. The recombinant plasmid pEFAKB1 was sequenced and introduced by electroporation into E. coli SCS110 (dam dcm mutant). The demethylated plasmid was then prepared from E. coli SCS110 and used for B. cereus ATCC 14579 electroporation.

Erythromycin-sensitive/kanamycin–resistant transformants were isolated by growing cells at 30°C on BHI agar plates containing erythromycin and kanamycin. Transformants were pooled, transferred into BHI broth, and incubated at a non-permissive temperature for plasmid replication (42°C). bc5181 disruption was verified by PCR with the primer pairs insKU1B/KSC4, insKU2B/BC5181R1H, and BC5181L1P/ywjAR1N (Table 2), as well as by DNA sequencing.

For overexpression experiments, an appropriate construct was prepared. bc5181 was amplified by PCR from B. cereus ATCC 14579 with the primers BC5181HindIII and BC5181SphI (Table 2). The 349 bp PCR product was cleaved with HindIII and SphI enzymes and ligated into the expression vector pDG148 (Stragier et al., 1988), previously digested with HindIII and SphI. The recombinant plasmid (pDGbc5181), carrying the bc5181 coding region under the transcriptional control of the inducible spac promoter (Pspac), was used to

transform B. cereus ATCC 14579 and B. subtilis 168 by electroporation and by chemical transformation respectively. Selection for 30µg kanamycin ml-1 _{or 5µg phleomycin ml}-1 resistance led to the isolation of MP25 and MP26 strain respectively. Pspac was induced by

adding IPTG (final concentration, 3 mM) to bacterial cultures. As control B. cereus ATCC 14579 was transformed with pDG148, thus obtaining MP28 strain.

Table 2. Primers used for PCR and RT-PCR amplification

Primer Sequence * Target gene/base pair coordinates† _{Restriction site} BC5181F2 5’-AAAAATCTCATATGGGAGGCTGT-3’ bc5181, from -28 to-6 None

BC5181R1 5’-GCTTTCGTAAGCGCCTGAG-3’ bc5181, from +137 to +156 None ywjAF2 5’GGAAAATGGAGGTAAGAT3’ ywjA, from -18 to -1 None insKU2B 5’-GGATCCAAATAGTGAATCATAAAAAAG-3’ insK, from +178 to + 205 BamHI

BC5181R1H 5’-AAGCTTAAAAAAATGCGA-3’ bc5181, from +212 to +229 HindIII

BC5181L1P 5’- CTGCAGTTTGCACCCATAATCG-3’ bc5181, from + 74 to +95 PstI

ywjAR1N 5’-CCATGGTGTGTTCTTATTCTCAG-3’ ywjA, from +1246 to +1269 NcoI

BC5181S1 5’-GCATGCCTCACTTTGATAC-3’ bc5181, from +303 to +316 SphI

BC5181H3 5’-AAGCTTATATGGGAAGGCTGTAC-3’ bc5181, from -22 to – 4 HindIII

insKU1B 5’-GGATCCAGTTAGCCAATCGTA-3’ insK, from +161 to +182 BamHI

KSC4 5’AAAGATCCGCGCGAGCTGTAT-3’ aphA3, from +1029 to +1050 None

*Restriction site is underlined

†_{Numbers refer to nucleotide position in the indicated gene, relative to the putative} translational initiation site.

3.4 Growth curves

Growth curves of all B. cereus strains were analysed in TrB, TrBG and BHIG at 37°C. The generation time (Tgen) was calculated as follows: Tgen=t/[3.3log(b/B)], where t is the time interval in minutes, b the number of bacteria at the end of the time interval, and B the number of bacteria at the beginning of the time interval.

3.5 Antimicrobial susceptibility

The antimicrobial susceptibility was evaluated with the E-test® method (based on antibiotic diffusion). The antibiotics used for E-test® method are benzylpenicillin (PG), tetracycline (TC), erythromycin (EM), ceftriaxone (TX), imipinem (IP), trimethoprim/sulfamethoxazole (PS), clindamycin (CM), rifampicin (RI), levofloxacin (LE), cefotaxime (CT), vancomycin (VA) and daptomycin (DPC) (AB Biodisk).

3.6 Biochemical profiles

Carbohydrate fermentation patterns were determined using API 50 CH/B strips following the

instructions provided by the manufacturer (bioMérieux, Lyon, France); test results were read

after 24h and 48h with the ATB Plus software (bioMérieux).

3.7 Treatment with peracetic acid

Experiments were performed essentially as previously described by Ceragioli et al. (2010). Briefly, B. cereus overnight cultures in BHI were used to inoculate 20 ml of BHI. The cultures were incubated at 30°C with shaking at 200 rpm for approximately 3h until the optical density at 600 nm (OD600) reached ~0.5 (~8 log CFU ml−1), which corresponded to the mid-exponential phase of growth. These cultures were subsequently treated with a mild (growth-attenuating) and lethal (i.e. a concentration inducing a 2-3 log reduction in the wild type strain)

concentrations of peracetic acid (PAA; Sigma-Aldrich) by adding a highly concentrated PAA

stock solution prepared in sterile demineralized water immediately before. An aliquot of each culture was collected before addition of the disinfectant (t0) and after 10 (t10), 30 (t30), and 60 (t60) min of exposure to the compound. The physiological response of the bacterial cells was investigated by enumeration of viable cells. Serial dilutions of cultures were made in peptone physiological salt solution (PPS) (1 g neutralized bacteriological peptone [Oxoid, England] l -1 _{and 8.5 g NaCl l}-1_{) and plated on BHI agar plates. Colonies were counted after 24 h of} incubation at 30°C.

3.8 Motility and chemotaxis assays

Phenotypic assays for swimming were performed on BHI fortified with 0.25% bacteriological agar (BHIM) as previously described by Senesi et al. with slight modifications (Senesi et al., 2002). 0.5 μl of an overnight culture was spotted onto the centre of BHIM, and growth halo diameters were measured after 6-8h of incubation at 37°C in a humidified chamber. Bacterial

motility was visualized by the hanging drop method by inspection of cultures obtained in BHI. Swimming motility was also evaluated under a phase-contrast microscope (BH-2; Olympus) by measuring the time of the smooth swimming phase between two consecutive tumblings. Isolated cells (n=50) were analysed in each sample for a total of three experiments performed on separate days. The extent of flagellation of cells grown in BHI was measured as previously described by subjecting purified extracellular flagellin to protein gel electrophoresis (Calvio et al., 2005). The intensity of the flagellin band, at different time intervals during growth, was measured by densitometric analysis using the Image Master 1D software (Pharmacia Biotech).

Chemotaxis assays was analysed on BHIM supplemented with 2 mM L-glutamine, as reviously described (Salvetti et al., 2007). Each chemotaxis assay was repeated three times on separate days and the chemotaxis index (CI) for each strain was calculated as follows: CI(Da-Dc))/Dc, where Da is the mean of growth halo diameters recorded on BHIM plates supplemented with L-glutamine and Dc the mean of those measured in BHIM without the attractant.

The ability of bacteria to swarm was tested on TrB, at 0.7% granulated agar as previously described (Salvetti et al., 2011). For each experiment, swarm plates were prepared fresh daily and allowed to sit at room temperature overnight before use. Plates were centrally inoculated with approx. 105 _{cells of B. cereus ATCC 14579 from a freshly grown overnight colony and} incubated at 37°C in a humidified chamber.

3.9 Insect infection

The contribution of BC5181 to B. cereus pathogenicity was assayed by comparing the killing effect of the wild-type, MP23, MP25 and MP28 B. cereus strains after injecting them separately into the hemocoel of Galleria mellonella larvae as previously described by Ramarao et al. (Ramarao et al., 2012). The surfaces of last-instar larvae weighing 180-250 mg were sterilized with 70% (vol/vol) ethanol. Bacterial cells previously grown in LB at 37°C under agitation were harvested at mid-exponential phase by centrifugation at 5,000 × g for 10 min at room temperature. A Hamilton syringe (Hamilton, Switzerland) was used to inject 10

a 26-gauge needle. Various doses (1 × 103 _{to 2.5 × 10}2 _{bacteria/larvae) were used to inject} groups of 20 larvae. Administered doses were determined by plating appropriate dilution of each dose on LB agar and counting the number of CFU formed after 16 h of incubation at 37°C. A control group of larvae was injected with PBS only. Infected larvae were kept at 37°C and mortality was recorded after 24 h. At least three independent experiments were performed for each strain. The 50% lethal dose (LD50; the dose by which 50% of the insects had died) was estimated using Probit analysis (Finney, 1972; Raymond et al., 1993).

3.10 Detection of phosphatidylcholine-preferring phospholipase C

(PC-PLC), proteases, haemolysins, and L

component of the haemolytic

enterotoxin (HBL)

For detection of virulence factors, whole or diluted supernatants of cells grown in TrB were used. PC-PLC and protease activity was measured as previously described (Ghelardi et al., 2002) by a gel-diffusion assay using respectively crude phosphatidylcholine (PC) and 1.5% Skim-Milk (Fluka, Sigma-Aldrich) as substrate (Wandersman et al., 1986). For PC-PLC quantification, different amounts of pure PC-PLC (Sigma-Aldrich) were used to obtain the standard calibration curve. The component L2 of HBL was detected in culture supernatants by immunoblot analysis. Nitrocellulose membranes (prepared as described above) were probed with rabbit polyclonal antibodies recognizing individual component L2, followed by incubation with a secondary antibody conjugated with horseradish peroxidase. The peroxidase activity was visualized by the diaminobenzidine colorimetric reaction in accordance with standard procedures (Sambrook et al., 1989). The lytic L2 component of (HBL) was also detected by using the commercial Bacillus cereus Enterotoxin-Reversed Passive Latex Agglutination kit (BCET-RPLA kit, Oxoid Ogdensburg, NY). General haemolysins were evaluated by loading wells incised in blood agar (Oxoid) with whole or diluted culture supernatants. Proteases, general haemolysins and the lytic component L2 of HBL were relatively quantified compared to wild type strain.

3.11 Protein samples and gel electrophoresis

Protein samples were prepared by growing bacterial cells to the late exponential growth phase in TrB at 200 r.p.m. at 37°C. Cultures were normalized to the same OD600 and aliquots of the resulting bacterial suspensions were pelleted by centrifugation at 10, 000 g. Culture

supernatants were collected and cell lysates prepared as previously described (Ghelardi et al., 2002). If necessary culture supernatants were concentrated with Microcon YM-10 filters (Millipore, Bedford, Mass). Protein concentration was determined by the Bradford method with BSA as a standard. Protein samples were suspended in NuPAGE LDS sample buffer (Invitrogen), heated at 70°C for 10 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and resulting gels were either silver stained or electrotransferred to nitrocellulose membranes for immunoblot analysis, using the XCell II Blot Module system (Invitrogen).

3.12 Biofilm formation

Biofilm formation on polystyrene microplates was measured as described previously (Wijman

et al., 2007) with some modifications. In brief, polystyrene microtiter plates (Becton

Dickinson, USA) were filled with 200μl LB medium, and for each strain, six wells were inoculated with 1.5% (vol/vol) overnight cultures, grown in LB. The plates were incubated at 30°C for 24 h, the medium was removed by gentle pipetting, and fresh medium was added to support biofilm development with new nutrients. After incubation for other 24 h, wells were gently washed three times with 200 μl of phosphate buffered saline, and subsequently biofilm cells were stained with 200 μl of 0.1% (wt/vol) crystal violet for 30 min. After these 30 min, the wells were washed twice with 200 μl sterile deionized water to remove unbound crystal violet. The remaining crystal violet was dissolved in 200 μl 96% ethanol, and the absorbance was measured at 570 nm with a microplate reader (model 550; Bio-Rad Laboratories S.r.l., Milano, Italy). Strains with optical density at 570 nm (OD570) values of 0.4, which is about two times the background signal, or higher were considered positive for biofilm formation. As control, two strains, B. cereus ATCC 14579, which is not able to form biofilm in LB after 48 h at 30°C and B. cereus ATCC 10987, which forms biofilm in LB after 48 h at 30°C, were

used. The average biofilm formation and standard deviation for six wells were calculated for each strain in the conditions tested.

3.13 Statistical analysis

Statistical analysis was performed with the Statistics software

(http://www.physics.csbsju.edu/stats/) and by Probit analysis (Finney, 1972; Raymond et al., 1993) for LD50. A P value <0.05 was considered significant. Values were expressed as the mean±SD from three independent experiments.

4. Results

---

4.1 bc5181: in silico analysis and gene expression

Systematic examination of the global transcriptional response of B. cereus ATCC 14579 following PAA treatment revealed an up-regulation in the expression of bc5181 (Ceragioli et

al., 2010). bc5181 is the last gene of a group of genes with the same orientation. The upstream

gene, ywjA, encodes a multidrug ABC transporter permease/ATP-binding protein. Downstream bc5181 and in the opposite orientation there is insK, a putative transposase encoding gene.bc5181 encodes a putative protein of 103 aa, having an estimated molecular

mass of 11.14 kDa and an isoelectric point of 4.6. Analysis of the amino acidic sequence with the TMpred program (www.ch.embnet.org) and signalIP (http://cbs.dtu.dk) showed that the protein encoded by bc5181 does not possess a transmembrane region and lacks the signal peptide cleavage site characteristic of secreted proteins. Therefore, BC5181 appears to be a cytoplasmic protein. As shown by sequence alignments and domain searches, BC5181 contains a domain with unknown function (DUF 74), which is shared by proteins belonging to the YbjQ-like family. Thus, BC5181 is considered a ybjQ-like encoding gene. PredictProtein (www.predictprotein.org) analysis revealed that the secondary structure of BC5181 is constituted by 36.9% α-helices, 37.7% strand, and 25.2% loops. Moreover, DisEMBL analysis (http://dis.embl.de) showed regions of structural variation.

In silico analysis of the B. cereus ATCC 14579 genome revealed the presence of two ORFs

(bc1012, bc1816) highly homologous to bc5181 (84.93% nt and 87.3% aa sequence identity for bc1012, 82.37% nt and 87.3% aa sequence identity for bc1816).

RT-PCR analysis on total RNA purified from B. cereus grown in TrB showed an amplification product of the predicted size with the primer pair BC5181F2/BC5181R1, designed on the sequence of bc5181, thus indicating expression of bc5181 in this growth condition (Fig. 5, lane 1). We also evaluated whether bc5181 was part of a transcriptional unit

also containing ywjA. No amplification product was obtained using the primer pair ywjAF2/BC5181R1 that was designed on the sequence of bc5181/ywjA. Therefore, bc5181 constitutes a monocistronic transcriptional unit (Fig. 5, lane 3).

Figure 5. Analysis of the expression of bc5181 in B. cereus ATCC 14579. Electrophoretic profiles of RT-PCR amplified products obtained on total RNA (line 1 and 3) and chromosomal DNA (line 2 and 4). Primers used in different reactions are shown at the top of the image. Lane M, molecular size standard.

4.2 bc5181 does not affect growth, biochemical profile and antibiotic

resistance

A bc5181 null mutant (MP23) was constructed by allelic exchange with a kanamycin resistance gene (Kmr_{). B. cereus was also transformed with the plasmid pDG148 and pDG148}

harbouring bc5181 under the IPTG-inducible Pspac transcriptional promoter (pDGbc5181), thus generating strain MP28 and MP25, respectively. The Pspac transcriptional promoter of

plasmid pDG148 has already been shown to be active at basic levels in the absence of inducer, but gene expression can be further stimulated by increasing IPTG concentration (Joseph et

al., 2001).

No differences in the growth curves and generation times were observed among the strains

grown in TrB, TrBG, and BHIG, with or without 3mM IPTG (Tgen ranging from 30.68 ± 2.9

to 35 ± 1.7 min in TrB, 27.54 ± 1.9 to 31.8 ± 2.4 min in TrBG, and 18.46 ± 2.2 to 20 ± 2.5 min in BHIG) (Fig. 6).

A

Figure 6. Growth curves of wild type, MP23, MP25 , MP25 with IPTG induction and MP28 strains in TrB (A), TrBG (B) and BHIG (C)

The biochemical profile, evaluated by using the API 50-CHB strip tests (bioMèrieux), revealed no difference in the ability to use carbohydrates among strains (Table 3).

Since an E. coli mutant lacking ybjQ was shown to be more susceptible to some antibiotics (Lui et al., 2010), we tested the susceptibility of all strains to different classes of drugs. No difference was observed between the susceptibility of MP23 and wild type strain, and between the susceptibility of MP25 and its control MP28 to penicillin (PG), tetracycline (TC), macrolide (EM), cephalosporins (CT, and TX), carbapenem (IP), lincosamide (CM), rifamycin (RI), quinolone (LE), glycopeptide (VA), lipopeptide (DPC) and trimethoprim PS (Table 4). The result obtained with the MP28 strains demonstrated that differences of susceptibility observed between the wild type and the MP25 was only due by the presence of the plasmid pDG148.

Property ATCC 14579 MP23 MP25 MP28 Glycerol - - - - Erythritol - - - - D-arabinose - - - - L-arabinose - - - - Ribose + + + + D-xylose - - - - L-xylose - - - - Adonitol - - - - β-Methyl D-xyloside - - - - Galactose - - - - Glucose + + + + Fructose + + + + Mannose - - - - Sorbose - - - - Rhamnose - - - - Dulcitol - - - - Inositol - - - - Mannitol - - - - Sorbitol - - - - α- Methyl -D-mannoside - - - - α-Methyl -D-glucoside - - - - N-acetyl glucosamine + + + + Amygdalin - - - - Arbutin + + + + Esculin + + + + Salicin + + + + Cellobiose + + + + Maltose + + + + Lactose - - - - Melibiose - - - - Saccharose + + + + Trehalose + + + + Inulin - - - - Melezitose - - - - Raffinose - - - - Starch + + + + Glycogen + + + + Xylitol - - - - Gentiobiose - - - - D-turanose - - - - D-lyxose - - - - D-tagatose - - - - D-fucose - - - - L-fucose - - - - D-arabitol - - - - L-arabitol - - - - Gluconate - - - - 2-Keto-gluconate - - - - 5-Keto-gluconate - - - -

Table 4: Antimicrobial susceptibility of B. cereus ATCC 14579 and its derivatives, MP23, MP25 and MP28 to antimicrobials by Etest ® method.

Antibiotic WT MP23 MP25 MP28 PG 16 16 >256 >256 TC 2 2 2 2 EM 0.47 0.47 0.64 0.64 TX 4 4 4 4 IP 0.94 0.94 0.125 0.125 PS >32 >32 3 2 CM 0.125 0.125 0.125 0.125 RI 0.5 0.5 0.38 0.38 LE 0.94 0.94 0.125 0.19 CT N.D N.D >32 >32 VA 1.5 1.5 2 2 DPC 0.5 0.5 3-4 3

4.3 Overexpression of bc5181 increases resistance to PAA treatment

According to Ceragioli et al. bc5181 is significantly up-regulated under lethal PAA stress (Ceragioli et al., 2010), suggesting a potential role for BC5181 in the response to this disinfectant. To test this hypothesis, the experiments described by Ceragioli et al. were reproduced with the strains ATCC 14579, MP23, MP25, and MP28. Mid-exponential phase cells were treated with two PAA concentrations and bacterial survival evaluated after 10, 30 and 60 min exposure. No significant difference was observed in the survival curves when the strains were treated with the mild PAA concentration (Fig. 7a). However, when the lethal concentration was used, a high resistance to PAA was observed in MP25. Indeed, MP25 survival was significantly increased compared to the wild-type at all-time points analysed (P≤0.01; Fig. 7b) in the absence of inducer and, more pronouncedly, following IPTG induction. The comparable survival curves obtained for MP28 and the wild type strain demonstrate that the increased resistance of MP25 to PAA is due to bc5181 overexpression. Unexpectedly, a significant increase in the resistance to the lethal PAA

concentration was also observed in the bc5181 null mutant compared to the wild type (P≤0.01).

Figure 7. Survival curves of mid-exponential phase (OD600 ~ 0.5) B. cereus ATCC 14579 (WT) (dark line and

white circle), MP23 (dark line and white triangle), MP25 without IPTG (grey line and black cross), MP25+IPTG (dark line and white square) and MP28 (dark line and white diamond), cells upon exposure to PAA treatment. Two different concentrations were selected, mild concentration (a) and lethal concentration (b). The dashed lines represent the counts derived from non-treated samples. The average of three independent experiments are shown and error bars indicate standard deviations.

4.4 bc5181 and motility

In order to investigate on the involvement of bc5181 in bacterial motility and chemotactic behaviour, we comparatively analysed swimming motility, chemotactic response and swarming ability of all strains.

Swimming assays on BHIM revealed no effect of bc5181 deletion on B. cereus motility, being 40.5 ± 3.4 mm and 35.0 ± 3.5 mm the diameters of migration halos of the wild type and MP23, respectively. In contrast, a significant decrease in motility halos (P≤0.001) was shown

at elevated bc5181 expression levels (MP25: 3.2 ± 0.5 mm; MP25 with IPTG-induction: 3.6

± 0.9 mm) (Fig. 8a). The reduced motility of MP25 was not due to lack of flagellar filaments, as shown by SDS-PAGE analysis of the extracellular flagellin collected from cells grown in liquid cultures (Fig. 8b). Microscopic examinations of bacteria by the hanging-drop method revealed that all strains alternated tumbling and smooth swimming. However, smooth swimming phases were significantly shorter in strain MP25 with (2.12 ± 0.6 s) or without IPTG addition (1.98 ± 0.4 s) than in the wild type (5.3 ± 0.9 s), MP23 (4.8 ± 1.0 s), and MP28 (4.7 ± 1.2 s) strains (P≤0.001). These results indicate that bc5181 overexpression causes a defect in swimming motility. Overexpression of bc5181 also caused a defect in chemotactic response (Fig. 8c). In fact, while MP23, MP28 and the wild type showed comparable chemotaxis indexes toward L-glutamine, the ability of MP25 to move in response to the chemoattractant was reduced, as shown by the low chemotaxis indexes calculated for this strain with or without IPTG induction.

B. cereus is able to move by swarming, a specialized form of flagellum-driven motility

that allows cells to co-ordinately migrate and colonize solid surfaces (Senesi et al., 2002; Ghelardi et al., 2007; Salvetti et al., 2011). To evaluate whether bc5181 deletion or overexpression could influence B. cereus swarming behaviour, a swarming assay originally described for strain ATCC 14579 was performed (Salvetti et al., 2011). As showed in Figure 10, among all strains only MP25 resulted to be impaired in producing swarming colonies on a solid medium (Fig. 8d).

These observations indicate that, while lack of bc5181 does not cause alterations in B.

cereus motility, the level of expression of bc5181 is critical in controlling swimming as well

Figure 8. Involvement of bc5181 in B. cereus motility and chemotaxis. (a) Swimming haloes of B. cereus ATCC 14579 (wt) MP23 (bc5181 null mutant), MP25 (over-expression of bc5181), and MP28 (MP25 control) on motility plates (BHIM and BHIGM). (b) SDS-PAGE analysis of total extracellular flagellin collected from bacteria grown in broth cultures (BHI) normalized to the same OD600. (c) diameters of motility haloes on BHI

(Dc), BHI supplemented with L-glutamine (Da). Chemotaxis indexes (CI) were calculated as described in Methods. (d) Colonies produced by swarming (ATCC 14579, MP23 and MP28) and non-swarming (MP25 with or 3mM IPTG induction) B. cereus strains on tryptone-NaCl containing 0.7% agar. Plates were photographed after 8h of incubation at 37°C.

4.5 Contribution of bc5181 to B. cereus pathogenicity and virulence

To investigate the effect of bc5181 deletion or overexpression on B. cereus pathogenicity, G.

mellonella larvae were used as an in vivo model of infection with the strains ATCC 14579,

MP23, MP25, and MP28. After 24 hours of infection with three doses of mid-log phase bacteria (2.5 × 102_{; 5.0 × 10}2_{; 1.0 × 10}3_{), the number of dead larvae was recorded (Fig. 9).}

No difference was observed in the mortality rates after injection of MP23, MP28 and the wild-type strain at different doses. Interestingly, a significant reduction in mortality was

observed when MP25 was inoculated at 2.5 × 102 _{and 5 × 10}2 _{bacteria/larva (P≤0.001). The}

50% lethal doses (LD50s) were assessed by Probit analysis and resulted to be 2.67 × 102 (95%

confidence limits between 2.25 × 102 _{and 3.17 × 10}2_{) bacteria/larva for the wild-type strain,}

2.32 × 102 _{(95% confidence limits between 1.82 × 10}2 _{and 2.96 × 10}2_{) bacteria/larva for}

MP23, 4.97 × 102 _{(95% confidence limits between 4.12 × 10}2 _{and 6.00 × 10}2_{) bacteria/larva}

for MP25, and 2.60 × 102 _{(95% confidence limits between 2.22 × 10}2 _{and 3.06 × 10}2₎

bacteria/larva for MP28 (Table 5). Therefore, an increase in bc5181 expression levels leads to a reduction in B. cereus pathogenicity in the G. mellonella model, with an almost two-fold increase in the LD50 of MP25 compared to the parental strain.

Figure 9. Effect of bc5181 expression on B. cereus virulence after intrahemocoelic injection in Galleria mellonella of B. cereus wild-type, MP23, MP25, and MP28 strains. Last-instar larvae were injected with three doses (2.5 × 102_{, 5.0 × 10}2_{, and 1.0 ×10}3_{) of mid-log phase bacteria of each strain. Mortality was evaluated after}

24 h of injection. Results are mean values of three independent experiments and bars indicate the standard errors. Asterisks (*) denote statistical significance (P<0.01).

Table 5. Letal Dose 50 (LD50) of the wild-type (WT) strain of B. cereus ATCC 14579 and its derivatives,

MP23, MP25 and MP28 to G. mellonella by Probit Analysis.

Some relevant virulence factors secreted by B. cereus were quantified in supernatants of late exponential phase cultures of all strains. As shown in Table 6, a substantial reduction in the amount of PC-PLC, proteases and general haemolysins was observed in MP25 compared to the wild type. This reduction was particularly evident when bc5181 expression was further increased by the addition of IPTG. Since MP28 behaved as the wild type, the observed reduction in the secretion of virulence factors in MP25 was only due to the higher bc5181 expression. Haemolysin BL secretion was not affected by bc5181 expression. MP23 strain presented a slight reduction of the virulence profile compared to wild type (Table 6).

These results indicate that alterations in bc5181 levels of expression affect virulence profile and pathogenicity of B. cereus.

Table 6. Quantification of secreted virulence factors

Strain PC-PLC∗∗∗∗ Lytic component L2 Proteases§ Haemolysis ‖‖‖‖ BCET-RPLA kit†††† Immunoblot with anti-L2 antibodies‡‡‡‡ ATCC 14579 13.4 ±±±± 0.53 +++ 1 100 100 MP23 6.64 ±±±± 0.34** ++ 0.5 75 75 MP25 1.8 ±±±± 0.2** +++ 1 50 37.5 MP25+IPTG (3mM) 1.3 ±±±± 0.2** +++ 1 25 37.5 MP28 13.56 ±±±± 0.8 +++ 1 100 100 ∗ ∗ ∗

∗ _{The amount of PC-PLC was expressed as mU mg-1 of total proteins in supernatant of culture.}

†_{The BCET-RPLA is specific for HBL detection. Results were scored as positive (+, ++, or +++) or negative} (-) according to the manufacturers’ directions.

‡+§+‖‖‖‖_{the wild type strain was used for relative quantification of Lytic component L}

2 with immunoblot with

anti-L2 antibodies, proteases and haemolysis.

**Statistical analysis of the C mean ratios for the five groups of strains was performed by t-Student (p<0.001) statistical test.