Lactic acid bacteria and their antimicrobial peptides : Induction ... - Helda

Lactic Acid Bacteria and Their Antimicrobial Peptides: Induction, Detection, Partial Characterization, and Their Potential Applications

HANAN T. ABBAS HILMI

Division of Microbiology Department of Food and Environmental Sciences Faculty of Agriculture and Forestry University of Helsinki

Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki

16/2010

LACTIC ACID BACTERIA AND THEIR ANTIMICROBIAL PEPTIDES: INDUCTION, DETECTION, PARTIAL CHARACTERIZATION, AND THEIR POTENTIAL APPLICATIONS

Hanan T. Abbas Hilmi

Department of Food and Environmental Sciences Division of Microbiology Faculty of Agriculture and Forestry University of Helsinki Finland

Academic Dissertation in Microbiology

To be presented, with permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in auditorium 1041, Viikki Biocenter, Viikinkaari 5, Helsinki, on June 18th at 12 o’clock noon. Helsinki 2010

Supervisor: Professor Per Saris Department of Food and Environmental Sciences University of Helsinki Finland

Reviewers:

Professor Seppo Salminen Department of Biochemistry and Food Functional Food Forum Faculty of Mathematics and Natural Sciences University of Turku Finland Professor Matti Karp Institute of Environmental Engineering and Biotechnology Tampere University of Technology Finland

Opponent: Professor Djamel Drider Nantes Atlantic College of Veterinary Medicine, Food Science and Engineering,ONIRIS-Nantes. France

Printed: Yliopistopaino 2010, Helsinki, Finland Layout: Timo Päivärinta ISSN: 1795-7079 ISBN: 978-952-10-6329-9 (paperback) ISBN: 978-952-10-6330-5 (PDF) email: [email protected]

To my beloved parents, husband, son and daughter

TABLE OF CONTENTS LIST OF ORIGINAL PUBLICATIONS THE AUTHOR’S CONTRIBUTION ABBREVIATIONS ABSTRACT INTRODUCTION .............................................................................................................. 1 1. LACTIC ACID BACTERIA ................................................................................... 1.1. Antimicrobial peptides produced by lactic acid bacteria ..................................... 1.2. Impact of LAB and their antimicrobial peptides in food safety and therapy ......... 2. NISIN ................................................................................................................... 2.1. Nisin biosynthesis ........................................................................................... 2.2. Regulation of nisin .......................................................................................... 2.3. Nisin secretion; transport and proteolytic processing .......................................... 2.4. Detection and quantification of nisin .................................................................

1 1 2 5 6 7 8 9

3. FLOW CYTOMETRY (FCM) AND FLUORESENCE-ACTIVATED CELL SORTING (FACS) OF BACTERIAL CELLS .............................................. 11 4. CHICKEN MICROBIOTA AND POTENTIAL OF LAB AND/OR THEIR ANTIMICROBIAL PEPTIDES IN CHICKEN FARMING ......................... 4.1. Alternatives to growth-promoting antibiotics (GPA) in poultry production ........ 4.1.1. Competitive enhancement techniques (CE) ........................................... 4.1.1.1. Competitive exclusion ............................................................. 4.1.1.2. Probiotics ............................................................................... 4.1.1.3. Prebiotics .............................................................................. 4.2. Campylobacter and chicken farming ............................................................... 4.2.1. Peptide bacteriocins ............................................................................. 4.2.2. Phage therapy ...................................................................................... 5. AIMS OF THE STUDY .......................................................................................

13 13 14 14 16 17 18 19 20 21 22 22 24 24 25 25 26 26 27 27 27 28 28 28

6. MATERIALS AND METHODS ........................................................................... 6.1. Strains and plasmids ..................................................................................... 6.2. Methods ....................................................................................................... 6.2.1. Northern hybridization analysis ............................................................ 6.2.2. Nisin bioassay ..................................................................................... 6.2.3. GFP and GFPuv based bioassays ............................................................ 6.2.4. Fluorescence-activated cell sorting ....................................................... 6.2.5. Chickens and diets ............................................................................... 6.2.6. Isolation and enumeration of crop bacteria ............................................ 6.2.7. Identification of the crop microbiota ..................................................... 6.2.8. Fatty acid analysis and FAME chromatography of L. reuteri strains ........ 6.2.9. Detection of antagonistic activity of L. salivarius strains against C. jejuni 6.2.10. Purification of the bacteriocin from bacterial culture ............................ 6.2.11. Solid phase extraction(SPE) ............................................................... 6.2.12. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) .............................................................. 29

7. RESULTS AND DISCUSSION ............................................................................ 7.1. Nisin induction studies (I, II) ......................................................................... 7.1.1. Knockout of the nisT gene in L. lactis caused induction of the PnisZ promoter ............................................................. 7.1.2. Nisin detection in the medium and the cytoplasm of L. lactis cells .......... 7.1.3. Activation of fully modified nisin precursor in the cytoplasm of L. lactis .......................................................................... 7.1.4. How does intracellular nisin induce the nisin promoters? ....................... 7.1.5. Isolation of natural mutants of the GFPuv nisin bioassay indicator L. lactis LAC275 cells by FACS(II) .......................... 7.2. Applied focus:secreening of bacteriocinogenic lab strains for future feeding trials in chicken(III,IV) ............................................ 7.2.1. Characterization of the crop microbiota(III) ........................................... 7.2.2. Characterization of L. salivarius bacteriocin which exhibit antagonistic activity against Campylobacter jejuni(IV) .......................... 8. CONCLUSIONS .................................................................................................

30 30 30 30 31 32 33 34 34

35 37 9. FUTURE PROSPECTS ........................................................................................ 38 10. ACKNOWLEDGEMENTS ................................................................................ 39 11. LITERATURE CITED ....................................................................................... 41

LIST OF ORIGINAL PAPERS This thesis is based on the following original papers, referred to in the text by their Roman numerals. I.

Hanan T. Abbas Hilmi, Kari Kylä-Nikkilä, Runar Ra and Per E. J. Saris. 2006. Nisin induction without secretion. Microbiology 152: 1489-1496.

II.

Hanan T. Abbas Hilmi, Kaisa Hakkila and Per E. J. Saris. 2009. Isolation of sensitive nisin-sensing GFPuv bioassay Lactococcus lactis strains using FACS. Biotechnol. Lett. 31: 119–122.

III. Hanan T. Abbas Hilmi, Anu Surakka, Juha Apajalahti and Per E. J. Saris. 2007. Identification of the most abundant Lactobacillus species in the crop of 1- and 5-weekold broiler chickens. Appl. Environ. Microbiol. 73: 7867-7873.

IV. Hanan T. Abbas Hilmi, and Per E.J. Saris. 2010. Characterization of Lactobacillus salivarius bacteriocin inhibitory to Campylobacter jejuni. Submitted to J. Appl. Microbiol. The original papers were reproduced with the kind permission of the copyright holders: Society for General Microbiology,American Society for Microbiology and Springer.

The author’s contribution Publication I Hanan Abbas Hilmi performed all the experimental work (excluding the Northern hybridization), interpreted the results and wrote the Paper in collaboration with the corresponding author. Publication II Hanan Abbas Hilmi performed all experimental work (excluding FCM SOFTWANAk), interpreted the results and wrote the article in collaboration with the corresponding author. Publication III Hanan Abbas Hilmi performed all the experimental work, (excluding the isolation and identification of Lactobacillus species, though the identifity of L. salivarius LAB47 was confirmed by Abbas Hilmi).Abbas Hilmi interpreted the results, and the diversity results were interpreted in collaboration with Anu Surakka and corresponding author. Abbas Hilmi wrote the article in collaboration with the corresponding author. Publication IV Hanan Abbas Hilmi designed the experiments, performed all experimental work, interpreted the results and wrote the article in collaboration with the corresponding author.

ABBREVIATIONS aa ABC Abu Dha Dhb AMP CE CFU DFM e. g. et al. etc. GALT GFP GFPuv GIT GPA GRAS HK IM IU kDa LAB Lan LB LBS MeLan MIC OD FACS FCM PCR PMSF RK SCFA SDS TCS QS

Amino acid ATP-binding cassette Aminobutyric acid Dihydroalanine Dihydrobutyrine Antimicrobial peptides Competitive exclusion Colony-forming unit Direct-fed microbials example gratia, for example et alii , and others et cetera, and so on Gut-associated lymphoid tissue Green fluorescent protein Ultraviolet variant of green fluorescent protein Gastrointestinal tract Growth promoting antibiotics Generally recognized as safe Histidine kinase Integral membrane International unit Kilodalton Lactic acid bacteria Lanthionine Luria Bertani medium Lactobacillus selective medium Methyl-lanthionine Minimum inhibitory concentration Optical density Fluorescence activated cell sorting Flow cytometry Polymerase chain reaction Phenylmethylsulfonyl fluoride Response regulator Short chain fatty acid Sodium dodecyl sulphate Two-component system Quorum sensing

ABSTRACT Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fluorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

Introduction

1. LACTIC ACID BACTERIA Lactic acid bacteria (LAB) constitute a diverse group of Gram-positive bacteria, characterized by some common morphological, metabolic and physiological traits. They are anaerobic bacteria, non-sporulating, acid tolerant and produce mainly lactic acid as an end product of carbohydrate fermentation. Lactic acid bacteria consist of a number of diverse genera which include both homofermenters and heterofermenters based on the end product of their fermentation.The homofermenters produce lactic acid as the major product of fermentation of glucose and include the genera Lactococcus, Streptococcus and Pediococcus. In contrast, the heterofermenters produce a number of products besides lactic acid, such as carbon dioxide, acetic acid, and ethanol from the fermentation of glucose and includes the genus Leuconostoc and a subgroup of the genus Lactobacillus, the Betabacteria (Jay 1986; Kandler et al. 1986; Schillinger and Lücke 1987). Lactic acid bacteria consist of a number of bacterial genera within the phylum Firmicutes. Recent taxonomic studies suggested that the LAB group includes the following genera; Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Lactosphaera, Leuconostoc, Melissococcus, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella (Ercolini et al. 2001; Holzapfel et al. 2001; Carr et al. 2002). Species of these genera can be found in the GIT of man and animal as well as in fermented food. LAB strains used as probiotics usually belong to species of the genera Lactobacillus, Enterococcus or Bifidobacterium. Gram-positive bacteria belonging to the phylum Actinobacteria, such as the genera Aerococcus, Microbacterium, Propionibacterium (Sneath and Holt 2001) and Bifidobacterium (Gibson and Fuller 2000; Holzapfel et al. 2001) also produce lactic acid. The core LAB genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus share a long history of safe usage in the processing of fermented foods. The antimicrobial effects and safety of LAB in food preservation is widely accepted (EFSA, 2005; de Vuyst and Leroy 2007; Sit and Vederas 2008). Their preservative effect is mainly due to the production of lactic acid and other organic acids which results in lowering of pH (Daeschel 1989). Preservation is enhanced by the production of other antimicrobial compounds, including hydrogen peroxide, CO2, diacetyl, acetaldehyde, and bacteriocins (Stiles and Hastings 1991; Klaenhammer 1988; 1993). In addition to the antimicrobial effects specific LAB also possess health promoting properties. Evidence from in vitro systems, animal models, and human clinical studies suggest that LAB function as immunomodulators and can enhance both specific and nonspecific immune responses (Ouwehand et al. 1998; 2002; O’Flaherty and Klaenhammer 2009) justifying their use as health promoting supplements or probiotics both for humans and animals.

1.1. Antimicrobial peptides produced by lab Lactic acid bacteria are capable of producing a wide range of ribosomally synthesized proteins and peptides which have antimicrobial activity to compete with other bacteria of the same species (narrow spectrum) or to counteract bacteria of other genera (broad spectrum) (Cotter et al. 2005). The first description of bacteriocin-mediated growth 1

Introduction

inhibition was reported 85 years ago, when antagonism between strains of Escherichia coli was first discovered (Gartia 1925). The inhibitory substances were called ‘colicins’, to reflect the producer organism, whereas gene-encoded antibacterial peptides produced by bacteria are now referred to as ‘bacteriocins’. Since their initial discovery in 1925, numerous bacteriocins have been isolated from LAB. Continuous discovery of new bacteriocins with novel features requires an updating of the classification of bacteriocins. Based on a recently proposed classification (Drider et al. 2006), there are three main classes of LAB-produced bacteriocins as shown in table (1). According to database BACTIBASE (Hammami et al. 2007), http://bactibase.pfbalab-tun.org/bacteriocinslist.php?start=1, a list of 180 entries, including 45 lanthioninecontaining bacteriocins (Class I), 55 non-lanthionine-containing bacteriocins (Class II) and 80 other unclassified entries are listed. Table 1. Classification of LAB bacteriocins according to Drider et al. 2006. Category

Subcategory

Class I Type A Lantibiotics, lanthionine containing Type Type B

Characteristics

Examples

elongated molecules: molecular mass 6.4 log reduction in 15-day-old chickens with a dose as low as 31.2 mg/kg feed. Both bacteriocins displayed strong broad spectrum antibacterial activity against food-borne pathogens Salmonella spp., E. coli O157:H7, Listeria spp., and Shigella spp. as well as showing potent anti-Campylobacter activity.

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Introduction

Clearly, anti-Campylobacter bacteriocin treatment is an effective strategy to reduce C. jejuni load in chickens. 4.2.2 Phage therapy Bacteriophages have unique advantages over antibiotics in that they are both self-replicating and self-limiting. The lytic capability and host specificity of phages also present an opportunity for using them to reduce the numbers of campylobacteria from animal sources and subsequently reducing cross contamination during processing. Campylobacter-specific bacteriophages have been isolated from various sources; pig manure, abattoir, sewage, and broiler chickens (Grawjewski et al. 1985; Atterbury et al. 2003; Goode et al. 2003). However, Hudson et al. (2005) showed that the activity of bacteriophages is maximal at the optimal growth temperature of their host which is 40°C in Campylobacter. Phages have been used to treat animal diseases such as Salmonella (Sklar et al. 2001) and Escherichia coli (Huff et al. 2003). Group II Campylobacter phage, CP220 (El-Shibiny et al. 2009), with a genome size of 197 kb was administered as a single 7-log PFU dose to both C. jejuni- and C. coli-colonized birds resulting in 2-log CFU/g decline in cecal Campylobacter counts after 48 h. Wagenaar et al. (2005) reported a10-fold reduction in Campylobacter colonization compared with control chickens by using Campylobacterspecific lytic phages. After an initial reduction, the camplylobacterial stabilized to levels of the untreated control chickens. Phages are unlikely to be applicable for the reduction of enteric pathogens in the intestine of poultry on rearing farms, since fecal shedding of both pathogen and phages would rapidly lead to resistance. Phage therapy against campylobacteria in chickens is not yet commercially available. Using a phage to control Campylobacter may not meet with public acceptance unless it is successfully implicated and comprehensively presented (Atterbury et al. 2003). Communication with the public is essential, and public acceptance may be as difficult to obtain as with genetically modified organism or their products. Leisner et al. (2005) described the obstacles for the introductionof pasteurization and starter cultures in the late 19th century to indicate that the gap between industry, consumers and pressure groups is not an entirely new issue.

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Aims of the study

5. AIMS OF THE STUDY The consumer demand for fresh, less processed food has increased the demand for natural food preservatives. Reducing the risk of human exposure to food-borne pathogens has a significant impact on food safety and public health. Thus, the goal of this study was to characterize the functions of nisin in different ecosystems knowing that nisin is a natural food preservative and has a capacity to kill food-borne pathogens, and in addition to find new bacteriocins for inhibition of campylobacteria. The detailed objectives are: 1. To study why the knockout of the NisT mutant caused induction of the nisZ promoter without addition of extracellular nisin. 2. To isolate natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fluorescence-activated cell sorting FACS. 3. To analyze the composition of the chicken crop microbiota and study the nisin resistance of the dominate crop species. 4. To isolate crop lactobacilli inhibiting Campylobacter jejuni and to characterize the inhibitory substance.

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Materials and methods

6. MATERIALS AND METHODS 6.1 Strains and plasmids The bacterial strains used in this study are shown in Table 1, plasmids in Table 2 and primers in Table 3. The methods used in this study are indicated in Table 4, but are described in more detail in the Materials and Methods sections of the Publications I- IV. All strains used are deposited in the Saris laboratory culture collection (Helsinki, Finland) and some strains also in the HAMBI culture collection. Table 1. Strains used in this study Strain

Relevant properties

Lactococcus lactis ssp. lactis N8 Wild-type nisin producer Non- nisin producer L. lactis ssp. lactis NZ9800 nisRK genes L. lactis ssp. lactis NZ9000 ∆nisT L. lactis LAC46 ∆nisB L. lactis LAC53 ∆nisC L. lactis LAC104 ∆nisZ L. lactis LAC67 LAC46/pLEB384 L. lactis LAC85 LAC53/pLEB384 L. lactis LAC109 LAC104/pLEB384 L. lactis LAC108 Host strain for constructed L. lactis MG1614 plasmids Fully modified prenisin L. lactisMG1614/pNZ9111 Indicator strain of GFP bioassay L. lactis LAC240 Indicator strain of GFPuv bioassay L. lactis LAC275 Natural mutant of LAC275 L. lactis LAC344 Natural mutant of LAC275 L. lactis LAC345 Resistant > 500 IU nisin ml-1 Lactobacillus reuteri HAN1-HAN19 Sensitive ≤ 50 IU nisin ml-1 L. reuteri HAN20-HAN41 Sensitive 50-500 IU nisin ml-1 L. reuteri HAN42-HAN88 Crop isolates L. reuteri HAN89-HAN98 Lactobacillus reuteri CHCC1956 Type strain for FAME analysis

Used in

Reference/source

I I III I I I I I I I I

Valio Ltd, Graeffe et al.1991 NIZO, Kuipers et al. 1993 NIZO, Kuipers et al. 1998 Ra et al. 1996 Qiao et al. 1996 Koponen et al. 2002 Qiao et al. 1996 This study This study This study Gasson 1984

I I I,III II II III

Kuipers et al. 1995 Reunanen and Saris 2003 Hakovirta et al. 2006 This study This study This study

III III III III

L. reuteri ATCC 55730

Type strain for FAME analysis

III

L. crispatus HAN1-HAN56 Lactobacillus salivarius LAB47 L. salivarius LAB48-LAB86 L. salivarius NRRLB-30514 Lactobacillus acidophilus/ johnsonii HAN156-HAN170 Lactobacillus sp. oral clone Lactobacillus sp. strain CLE-4 L. gallinarum HAN171 L. helveticus HAN172 L. pentosus HAN173 Pediococcus acidilactici HAN174-180 Micrococcus luteus A1NCIMB8166

Crop isolates Salivaricin 47 producer Crop isolates Bacteriocin OR-7 producer Crop isolates

III III,IV IV IV III

This study This study This study Christian Hansen Culture Collection, Denmark American Type Culture Collection This study This study This study Stern et al. 2006 This study

Crop isolate Crop isolate Crop isolate Crop isolate Crop isolate Crop isolates

III III III III III III

This study This study This study This study This study This study

Nisin- sensitive indicator strain

I

Campylobacter jejuni SAA553560

Indicator strains for detection of antagonistic activity of L. salivarius strains

IV

National Collection of Industrial and Marine Bacteria, UK. This study

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Table 2. Plasmids used in this study. Plasmid

Antibiotic resistance pLEB384 Ampr, Ermr pKTH1984 Camr, Ermr pLEB651 Camr

Relevant properties lactococcal expression plasmid, P45 source for nisZ probe lactococcal expression plasmid, PnisA

Used in I I II

Reference Qiao et al. 1996 Graeffe et al. 1991 Hakovirta et al. 2006

Amp = ampicillin, Cam = chloramphenicol, r = resistance Table 3. Sequences of PCR primers used in this study. Primer name

Sequence 5’→ 3’

Used in Original reference

pA(forward primer) pE (reverse primer) COLl (C. coli -specific primer) COL2 (C. coli -specific primer) JUN3 (C. jejuni -specific primer) JUN4 (C. jejuni -specific primer)

AGAGTTTGATCCTGGCTCAG CCGTCAATTCCTTTGAGTTT AGGCA AGG GAG CCT l T A ATC

III III IV

Edwards et al.1989 Edwards et al.1989 Vandamme et al.1997

TAT CCC TAT CTA CAA ATT CGC

IV

Vandamme et al.1997

CA TCT TCC CTA GTC AAG CCT

IV

Vandamme et al.1997

AAG ATA TGGCTC TAG CAA GAC IV

Vandamme et al.199

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6.2 Methods A detailed description of the methods is presented in the original publications I-IV. A brief summary and some additional information are presented in the following chapters. Table 4. Methods used in this study. Methods Strain isolation and/or growth L. lactis strains, M17G, growth medium (Oxoid, Unipath Ltd, England) containing 0.5% (w/v) glucose Lactobacillus strains, Modified Lactobacillus Selective Medium, mLBS (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) without acetic acid. For bacteriocin production L. salivarius LAB 47 were grown in minimal medium (Stern et al., 2006) Campylobacter jejuni Agar Base CM0689, Preston Campylobacter selective supplement SR0117 and lysed horse blood SR0048 (Oxoid Ltd, Finland) Induction & Identification Basic DNA techniques: DNA isolation, plasmid isolation, electrophoresis, PCR Northern hybridization Electrotransformation (Sambrook et al. 1989): E. coli and L. lactis (Holo and Nes 1989) Agar diffusion test (Tramer and Fowler 1964 ) Partial 16S rRNA gene sequencing Alignment and identification data base NCBI Blast Library (www.ncbi.nlm.nih.gov) Fatty acid analysis and FAME Chromatography, MIDI Inc., Newark, DE, USA. Bioscreen C (Labsystem, Finland) Nisin-induced GFP fluorescence (Reunanen and Saris 2003) Nisin-induced GFPuv fluorescence (Hakovirta et al. 2006 ) Fluorescence-activated cell sorting(FACS) Multiplex PCR assay (Vandamme et al. 1997) Well-diffusion assay (Schillinger and Luke 1989) Ammonium sulphate precipitation Solid –phase extraction (Phenomenex, Torrance, California, USA) Tricine SDS-PAGE Coomassie brilliant blue staining

Used in I, II III, IV IV IV

I-IV I I, III I III III III III I I,III II IV IV IV IV IV IV

6.2.1. Northern hybridization analysis L. lactis strains were grown to the exponential growth phase, collected by centrifugation and broken by sonication. RNA was isolated according to Ra and Saris (1995). Thirty micrograms of RNA and 3 mg RNA ladder (Gibco-BRL) were fractionated on 2% agarose gels containing formaldehyde. After electrophoresis the gel was soaked in water for 20 min and then for 1 h in transfer buffer (8 mM Na2HPO4, 17 mM NaH2PO4). RNA was transferred to Hybond N membrane with a Bio-Rad Trans-Blot cell overnight at 250 mA at 4 °C. After transfer, membranes were rinsed in transfer buffer and then incubated in a vacuum oven at 80 ◦C for 2 h. Membranes were prehybridized for 4 h at 57 ◦C (5x SSC, 5x Denhardt’s solution, 0.5% SDS and 200 mg denatured herring sperm DNA ml-1). After addition of the probe, hybridization was performed in the same buffer overnight at 57 ◦C. Membranes were washed for 10 min with 26 SSC/0.1% SDS at 57 ◦C, for 20 min with 16 SSC/0.1% SDS at 57 ◦C, for 20 min with 0.56 SSC/0.1% SDS at 57 ◦C and for 20 min at

24


65 ◦C, and finally with 0.16 SSC/0.1% SDS at 65 ◦C. Membranes were exposed to Kodak X-Omat 100 films at 270 ◦C overnight before developing. Labeling of 50 ng of probe was performed with the Multiprime labeling system (Amersham) according to the manufacturer’s instructions, in the presence of Amersham’s [32P] dCTP (3000 Ci mmol-1, 111 TBq mmol-1). Free nucleotides were separated from labeled DNA with Sephadex C-50 Nick columns (Pharmacia). The nisZ-specific DNA for the probe was madeby digestion of plasmid pLEB1984 (Graeffe et al. 1991) with SspI and SacI, generating a 227 bp fragment (the ends of the fragment are at bp 125–352 in the nisZ sequence at EMBL accession no. Z18947), which was isolated from a 0.8% agarose gel using a Magic PCR prep kit (Promega). 6.2.2. Nisin bioassay In order to detect the activity of nisin in the cytoplasm of lactococcal cells, L. lactis strains were grown to OD600 0.6. The cells were collected from 10 ml of culture by centrifugation and resuspended in 200 ml distilled water acidified to pH 2.5 with HCl. PMSF was used to irreversibly inhibit serine proteases NisP and trypsin at a final concentration of 10 mg ml-1 when needed. For isolation of intracellular nisin the cells were frozen by liquid nitrogen and thawed several times prior to sonication. Cytoplasm of LAC85 cells was also isolated without addition of PMSF for comparison. After sonication using a Labsonic U (Labsystems), the particulate fraction was removed by centrifugation at 14 000 g for 10 min and the supernatant was further filtered through a membrane with a cut-off of 0.2 mm. Antibacterial activity of nisin was determined as growth inhibition zones on Luria–Bertani soft agar plates inoculated with M. luteus. The indicator strain M. luteus was grown to OD600 0.7 and 10 μl of the culture was added to 3 ml Luria–Bertani soft agar. Three μl of the samples (both cells and supernatant for each strain) to be tested was applied as a droplet on the agar surface and allowed to dry. Plates were incubated overnight at 37 ◦C. The samples (both cells and supernatant) were heated at 80 ◦C for 10 min and stored frozen prior to the GFP bioassay. 6.2.3. GFP and GFPuv based nisin bioassays Two highly sensitive nisin bioassays were performed in this study as described by Reunanen and Saris (2003) and Hakovirta et al. (2006). The indicator strain LAC240 was used in the first assay and LAC275 in the latter. When LAC275 was used in the GFP bioassay the freezing step was omitted, chloramphenicol (10 mg ml-1) was added to the growth medium instead of erythromycin, less nisin was used for the standard curve, and the excitation and emission filters were different. Cells of the two indicator strains were inoculated 1 : 100 in M17 (Oxoid) containing 0.5% (w/v) glucose, 0.1% (w/v) Tween 80; when needed, 5 mg ml-1 erythromycin or 10 mg ml-1 chloramphenicol was added. The indicator strain inoculum was divided into aliquots of 175 ml on a microplate, then 50 ml aliquots of the samples were applied and the nisin standards (Sigma) were prepared so that the concentration of nisin in the culture medium ranged from 2.5 to 125 ng ml-1 for LAC240 and from 10 to 90 pg ml-1 for LAC275. The bacterial suspensions were divided into 225 ml aliquots on a microtitre plate, and incubated overnight at 30 ◦C. Before the fluorescence measurements 175 ml of each supernatant was removed. The maturation of newly synthesized GFP

25


molecules is a temperature-dependent process proceeding faster at low temperature (Lim et al. 1995). Thus, the incubated LAC240 cells were frozen for 30 min at 220 ◦C and thawed at room temperature prior to the detection of fluorescence. Cells of LAC275 did not require freezing for correct folding due to mutations in the gfp gene (Crameri et al. 1996). Green protein fluorescence was detected in terms of relative fluorescence units (RFU) with a Fluoroscan Ascent 374 fluorometer using the Ascent software (version 2.4.2; Labsystems). The excitation and emissions filters were 485 and 538 nm for LAC240, and 373 and 583 nm for LAC275. Growth was measured by determining the OD600 with an UltrospecII spectrophotometer (Pharmacia LKB). 6.2.4. Fluorescence-activated cell sorting FACSVantage flow cytometer SE (BD Biosciences) was used to sort cells of the L. lactis LAC275 strain at the Cell Imaging Core Facility Turku in Finland. Prior to sorting, cells of LAC275 were grown for 24 h at 30◦C with 10 pg nisin ml-1 for induction of GFPuv. After growth cells were diluted 1 : 1000 in PBS (0.15 M NaCl, 0.01 M sodium/ potassium phosphate buffer, pH 7.2) and analyzed with FACS using 488 nm argon laser for excitation and fluorescence was detected through 530/30 nm band-pass filter. Forward and side scatters and fluorescence data were collected using logarithmic amplification. About 2,000 events/s were analyzed with WinMDI program (version 2.8, Joseph Trotter http//facs.scripps.edu/). The sorted putative mutant L. lactis LAC275 cells were cultivated on plates overnight. Single colonies were separately tested by GFPuv nisin bioassay (Hakovirta et al. 2006) to analyze any change in nisin induction of the sorted LAC275 isolates. GFPuv fluorescence was measured as relative fluorescence units (RFU) with the Fluoroscan Ascent 374 fluorometer with Ascent software, version 2.4.2 (Labsystems). 6.2.5. Chickens and diets One- and 5-week-old broiler chickens with the same genetic background originating from the same hatchery (Broilertalo, Eura, Finland) were obtained from four Finnish farms (designated farm 1 [F1], in Eurajoki, F2, in Halikko, F3, in Lemu, and F4, in Marttila). The killing and sampling of birds were carried out under the Finnish law and statute on animal experiments and followed the ethical principles of the University of Helsinki. The chickens were fed with two brands (diet A and diet B) of wheat-based diets from two different commercial feed manufacturers, diet A was obtained from Suomen Rehu Ltd. (Espoo, Finland), and diet B was obtained from Raisio Feed Ltd. (Raisio, Finland), with both feeds providing two different wheat-based diets according to the growth phases of the chickens. Starter diets (including, at day 1, Broilact [Orion Corporation, Turku, Finland]) were fed during the first 2 weeks, followed by feeding with the grower diets. Broilact is a competitive exclusion preparation developed from the cecum content of chickens. Eight different lactobacilli have been isolated from the original inoculum, with one identified as being L. plantarum, one as being L. salivarius, and six as being obligate anaerobes without identification to the species level (Orion Corporation). The main difference between diet A and diet B was that diet A was supplemented with salinomycin (47 mg kg of feed-1) and diet B was supplemented with naracin (50 mg kg of feed-1) coccidiostat during the whole feeding period. Otherwise, the diets were very similar, 26


as the differences in dry matter (6.7 and 5.6% in diet A versus 6.3 and 5.9% in diet B), total fat (7.5 and 7.1% in diet A versus 6.7 and 6.3% in diet B), crude protein (20% in diet A versus 22 and 20% in diet B), and fiber (3 and 3.5% in diet A versus 3.6 and 3.7% in diet B) contents were minor. 6.2.6. Isolation and enumeration of crop bacteria Ten chickens, at 1 and 5 weeks of age, were randomly selected from each flock 2 h after feeding and humanely killed by cervical dislocation. Whole crops were removed from the carcasses and transported on ice to the laboratory. The crop (n=10) contents were pooled and homogenized in LBS medium (1:10) in a Stomacher laboratory blender (SewardMedical, London, United Kingdom) for 2 min. The crop homogenate was dilutedin LBS broth and cultivated by spread plating and pour plating onto LBS agar. The plates were incubated at 41.5°C for 48 h aerobically and anaerobically. For isolation of the crop bacteria located in the vicinity of or associated with the crop epithelium, a 1-cm2 piece from each of the 10 chicken crops (after gravity removal of the digesta and visual inspection to verify a clean mucosa) was cut, placed with the internal sides towards the surface of the LBS agar plates, and incubated overnight at 41.5°C. Before cutting the piece from the crop, the crop was opened with a knife, and the crop digesta was shaken away. The crop digesta dropped easily away from the mucosa, as it was almost solid, revealing the inner epithelium of the crop as being very clean by visual inspection. Bacteria from the obtained lawns of the 10 crop epithelium pieces were resuspended, pooled, and diluted in LBS broth, followed by plating onto LBS agar in order to obtain single colonies. The lactobacilli of the corresponding pooled crop contents were isolated as described above. Colonies were randomly selected and grown to pure cultures before DNA isolation. 6.2.7. Identification of the crop microbiota Identification of the crop isolates was based on partial 16S rRNA gene sequencing. To obtain a partial rRNA gene sequence, purified chromosomal DNA (van der Meer et al. 1993) from the isolates served as a template in PCR. The amplified area was defined by universal primers pA (5’-AGA GTT TGA TCC TGG CTC AG-3’) and pE (5’-CCGTCA ATT CCT TTG AGT TT-3’), which hybridize to the 16S rRNA gene at nucleotides 8 to 28 and 928 to 908 in Escherichia coli (Edwards et al. 1989). The PCR started with heating for 3 min at 94°C, followed by annealing for 1 min at 53°C and extension for 90 s at 72°C and by 30 cycles of PCR (consisting of 45 s at 94°C, 60 s at 53°C, and 90 s at 72°C) and ending the last cycle with storage at 4°C. One strand of the amplified 900-bp fragments was sequenced by the Synthesis and Sequencing Laboratory (Institute of Biotechnology, Helsinki, Finland). Strains with 16S rRNA genes exhibiting a sequence homology of 97% were regarded as belonging to the same species (Drancourt et al. 2000). The sequences obtained were compared against the National Center for Biotechnology Information genome BLAST library (version 2.2.8; http://www.ncbi.nlm.nih.gov/BLAST/). 6.2.8. Fatty acid analysis and FAME chromatography of L. reuteri strains The diversity of L. reuteri isolates was analyzed by gas chromatographic analysis of the whole-cell fatty acids. The extraction and derivatization method was done as described 27


previously by Miller (1982) and according to the protocol of the Sherlock microbial identification system (MIDI Inc.). Briefly, approximately 40 mg (wet weight) of cells was scraped from the surface of the agar and transferred into a tube with a Teflon-lined cap. Cells were saponified by heating the cells at 100°C/30 min following the addition of 1 ml of 15% NaOH in 50% aqueous methanol. The hydrolysate was cooled, 2 ml of methanolic HCl was added, and the mixture was heated at 80°C for 10 min. The methylated fatty acids were quickly cooled and extracted through the addition of 1.25 ml of hexane-methyl-tert-butyl ether (1:1, vol/vol) with end-end mixing. The phases were allowed to separate, the lower aqueous layer was removed, and 3 ml of dilute NaOH was added to the remaining organic layer. To further clarify the phase interface, saturated NaCl was added. Approximately twothirds of the organic layer (containing FAMEs) was transferred to a septum-capped vial for analysis. MIDI gas chromatography runs were calibrated against a standard mixture of known fatty acids provided by MIDI. Detected sample peaks were named by interpolation of the retention time using the equivalent chain length method. Peaks that did not display equivalent chains were unnamed. Two microliters of the fatty acid methyl esters was then analyzed using a 5890 A gas chromatograph fitted with a 5% phenyl methyl silicone capillary column, a flame ionization detector, an automatic sampler, and an integrator (Agilent Technologies, Diegem, Belgium) with the Microbial Identification system. 6.2.9. Detection of antagonistic activity of L. salivarius against C. jejuni L. salivarius cell-free supernatants (CFS) were obtained by centrifuging the cultures at 12 000 x g for 10 min, collecting the supernatants, which were adjusted to pH 6.5 with 1M NaOH, and filtered through a 0.22 μm filter (Millipore). Inhibitory activity from hydrogen peroxide was avoided by the addition of catalase (5 mg ml-1). A well diffusion assay (Schillinger and Luke 1989) was performed by using 15 ml Campylobacter selective medium (Oxoid, Finland) overlaid with 1% of an overnight culture of the indicator strain mixed with 5 ml of soft agar. Wells of 5 mm in diameter were cut into these agar plates and 25 μ1 of CFS of the potential producer strain was placed into well. The plates were incubated for 24 to 48 h at 37°C in a microaerophilic atmosphere. Activity was assessed by measuring the diameter of the zone of inhibition formed around the CFSs loaded wells. 6.2.10. Purification of the bacteriocin from the bacterial culture A cell-free solution (CFS) was obtained by centrifuging the 1 L -culture at 12 000 x g for 10 min. The pH of the CFS was then adjusted to 6.5 with 1N NaOH. The proteins of 500 ml CFS was precipitated with ammonium sulphate (45% saturation) overnight at 4°C with gentle stirring and centrifuged at 10 000 g for 30 min. The precipitate fraction was resuspended in 0.5 ml sodium phosphate buffer pH 7.2. 6.2.11. Solid phase extraction (SPE) Twenty μl of phosphoric acid was added to the resuspended fraction from the ammonium precipitation and loaded on strata-X (1 g/1 ml) column (Phenomenex, Torrance, California, USA). Then, the column was washed with 10% acetonitrile in 0.1% trifluoroacetic acid (TFA). Polypeptides were eluted from the column using acetonitrile/water (80:20 v/v). The eluted fraction (0.4 ml) was assayed for antimicrobial activity. 28


6.2.12. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) In order to determine which polypeptide in the elution fraction had anti-camplylobacterial activity a 15 μl sample was subjected to16.5% Tris-tricine-SDS-PAGE. After electrophoresis at 100 mA for 3 h, the gel was fixed with a solution containing 15% ethanol and 1% acetic acid. The gel was then washed with distilled water for 4 h. The gel was stained with a solution containing 0.15% Coomassie brilliant blue R-250 (Bio-Rad) in 40% ethanol and 7% acetic acid. Destaining was done with 10% acetic acid 3 times for 15 min. This gel with visible bands was further washed with phosphate-buffered saline (pH 7.2) for 1.5 h and then with deionized water for 3 h for the C. jejuni inhibition assay. To measure bacteriocin activity, the washed gel was placed onto semisolid Brucella agar (0.75% agar) overlaid with soft agar seeded with cells of C. jejuni SAA554, 555, 557 or 559 (Bhunia et al. 1987). Plates were incubated at 42°C for 48 h under a microaerobic atmosphere and inhibitory zones around the polypeptide bands were identified.

29

Results and discussion

7. RESULTS AND DISCUSSION 7.1. Nisin induction studies (I, II) 7.1.1 The knockout of the NisT mutant L. lactis caused induction of the PnisZ promoter (I) The regulation of nisin production by specific strains of Lactococcus lactis involves three genetically linked components: a secreted peptide pheromone, a histidine protein kinase (HPK) and a response regulator (RR) (Kleerebezem et al. 1997). The transmembrane HPK NisK senses nisin as signal, and activates the intracellular RR NisR, and subsequently results in activation of the nisin promoters, creating an autoinduction loop (Kuipers et al. 1995; de Ruyter et al. 1996; Qiao et al. 1996). Previously, Qiao and Saris (1996) reported that a L. lactis mutant with a deletion in the nisT gene did not secrete nisin and nisin could be detected upon lysis of these cells. Furthermore, complementation of this strain with a NisT encoding plasmid was sufficient to restore the nisin secreting phenotype. However, in that study nisin was detected from cytoplasmic fraction of the NisT mutant strain, but induction of nisin promoters by intracellularly accumulated nisin was not investigated. In this study, we present evidence of a novel, nisin induction of the nisZ promoter, which functions without secretion of nisin. In (I) the plasmid pLEB384, which has the nisin structural gene under the control of the nisininducible PnisZ and the constitutive P45 promoters, was transformed into NisT mutant LAC46 in an attempt to achieve greater prenisin production inside the cells (LAC46/pLEB384 = LAC85). Northern analysis of LAC85 with a nisZ probe showed that the PnisZ promoter was activated without addition of extracellular nisin. Compared to LAC109 (the nisB mutant) and LAC108 (the nisC mutant), which contain unmodified nisin precursor and dehydrated nisin precursor (Koponen et al. 2002), LAC85 cells were the only cells having active nisin in the cytoplasm and the PnisZ promoter activated without external nisin addition. Since nisin can be found inside LAC85 cells (I), it seems unlikely that the induction of the PnisZ promoter observed in these cells could have been due to intracellular production of nisin with the leader still attached, transported by some other transport protein followed by cleavage by NisP. This suggests that nisin can activate the nisin-inducible PnisZ promoter from inside the cells. This promoter is normally activated via the two-component regulatory proteins NisRK, but that requires external nisin (Kuipers et al. 1995). Cell lysing in a culture of LAC85 would release the intracellular nisin and may result in nisin induction of the neighboring cells. A sensitive nisin detection method was needed to detect if lysis of the cells could release enough nisin for the neighboring cells to get induced or if another explanation is needed for the induced state of the LAB85 cells. 7.1.2. Nisin detection in the medium and the cytoplasm of L. lactis cells To avoid the possibility that nisZ promoter activation was due to cell lysis leading to the release of nisin to the medium, and subsequently induction of intact cells, we determined the nisin content in cultures of the NisT mutant LAC85 using a sensitive nisin bioassay (Hakovirta et al. 2006). Cells of L. lactis LAC275 containing the nisin-inducible GFPuv gene were grown together with the growth supernatant of strain LAC85. The growth supernatant of LAC85 contained less than 10 pg nisin ml-1 (the detection limit of the assay) as the

30


supernatant was not able to induce any fluorescence in the cells of LAC275. To ensure that the LAC85 cells do not release nisin to the growth supernatant for other cells to be induced, LAC85 cells were grown together with LAC275 cells. No GFP-related fluorescence was obtained from the cell mixtures. These results indicated that the induced state of LAC85 cells do not arise from nisin in the media released by lysed LAC85 cells. Clearly, LAC85 cells are not inducing other cells in the surrounding as the co-growth with LAC275 should else have yielded induced LAC275 cells. All these results suggest that LAC85 cells can only induce themselves. In 1996 when Qiao and Saris first discovered that in a NisT mutant intracellular nisin accumulates in the cytoplasm without the leader (later also shown by van den Bergh et al., 2008) the possibility for leader processing by extracellular NisP after cell breakage was not excluded. Therefore, to verify whether the nisin activity detected from the isolated cytoplasm of LAC85 was formed before or after the breakage of the cells we added the serine protease inhibitor PMSF to the LAC85 cells before breakage of the cells. PMSF should be capable of inhibiting the NisP as it is a serine protease (van der Meer et al. 1993) anchored on the outside of the cells. We showed that PMSF could inhibit the NisP activity of intact LAC85 cells. No nisin activity was detected in LAC85 cells incubated together with PMSF and the fully modified nisin precursor still having the leader attached. To verify that the LAC85 cells contained active nisin and not prenisin with the leader still attached, the cells were broken in the presence of PMSF, to avoid the possibility that breakage of the cells would release nisin precursor with the leader to the NisP protease present on the surface of the cells, resulting in false positive results. Nisin from the cytoplasm of LAC85 isolated in the presence of PMSF was quantified using the nisin-induced green fluorescence bioassay (Reunanen and Saris 2003). The results showed that the cytoplasm of LAC85 cells in the logarithmic growth phase (OD600 = 0.5) contained nisin at a concentration of 6.2±1.6 ng nisin ml-1. Nisin could also be isolated from the cytoplasm of LAC85 cells that had been grown in the presence of PMSF. This showed that nisin with the leader attached and for some reason crossing the cytoplasmic membrane without NisT and potentially cleaved by NisP resulting in induction is not an explanation for the induced state of the LAC85 cells. In addition, the nisin activity of the cytoplasm of LAC85 isolated with or without PMSF was analyzed using nisin-sensitive M. luteus as an indicator. If the cytoplasm of LAC85 contained fully modified nisin precursor with the leader still attached, then isolation of the cytoplasm without PMSF would result in cleavage of the leader, resulting in increased inhibition compared to cytoplasm isolated with PMSF included. The nisin activity of the cytoplasm of LAC85 cells +/- PMSF during isolation was in the same range (I, Fig. 2b) showing further that when nisin transport is blocked active nisin is accumulated in the cytoplasm. This has also been verified in another NisT mutant (van Saparoea et al. 2008). 7.1.3. Activation of fully modified nisin precursor in the cytoplasm of L. lactis The fact that the cytoplasm of LAC85 contains active nisin provides evidence that L. lactis does have intracellular proteolytic activity capable of degrading the leader from the fully modified nisin precursor. We have not identified the protease capable of nisin leader cleavage from the genome of the sequenced L. lactis subsp. lactis IL1403 strain (Bolotin et al. 2001), but the gene content of different strains may differ (Kok et al. 2005). Proteins have a certain half-life in cells and several proteolytic systems for this protein turnover are

31


present in all living cells. The leader has an elongated α-helical structure and could fit into the proteasome and be degraded, whereas the rest of the molecule has a more rigid and compact structure and may not fit into the proteasome (Groll and Clausen 2003). 7.1.4. How does intracellular nisin induce the nisin promoters? One key question is how does the intracellular nisin of LAC85 cells, deficient in nisinspecific secretion capacity, reach the externally located signal recognition domain of the NisK? One possibility was that the LAC85 cells had lysed, and released nisin to the intact cells resulting in induction of PnisZ . This possibility was excluded as nisin could not be detected in the culture supernatant using the sensitive nisin detection and quantification system (Hakovirta et al. 2006), which could detect nisin in concentrations as low as 10 pg nisin ml -1. Moreover, if the LAC85 cells released less than 10 pg nisin ml -1, then this concentration would not have been sufficient to induce the nisZ promoter as strongly as judged from the Northern blots (I, Fig. 1). The possibility that nisin could to some extent be translocated to the pseudoperiplasmic space by transporter other than the NisT transporter cannot be excluded. In the pseudoperiplasmic space nisin is either adsorbed to the membrane surface or to the cell

Figure 6. Nisin and signal recognition possibilities by NisK. A) extracellular nisin recognition (Kuipers et al. 1995). B) transport of nisin by ABC transporter NisFEG. C, D, and E are suggested locations for nisin: C) nisin adsorbs to the membrane; D) nisin inserts to membrane and presents the C-terminus of the molecule into the pseudoperiplasmic space for the NisK signal recognition domain ;E) nisin is in the pseudoperiplasmic space. 32


wall or ends up in the growth medium by diffusion or active transport. Stein et al. (2003) showed that the nisin ABC transporter NisFEG acts as a nisin exporter that expels nisin molecules from cells into surface. It seems that nisin in LAC85 is situated such that NisFEG cannot expel nisin to the medium as nisin could not be detected in the culture medium of LAC85 cells. Therefore, we suggest that in LAC85 cells, a fraction of the intracellular nisin is inserted into the membrane and some part of nisin is extruded to the outer side of the membrane and there recognized by NisK. Peptide pheromones seem to use their amphiphilic C-terminal domain to bind to the histidine protein kinase, possibly at membrane level, in a relative non-specific fashion (van Belkum et al. (2007). The weak interaction is greatly improved by the N-terminal domain of the peptide pheromone that is highly specific for the cognate receptor. This interaction will enable the peptide pheromone to activate bacteriocin production at less than nanomolar levels. van Belkum et al. (2007) reported that the C-terminal domain of the peptide LV17A and enterocins A and B pheromones interact relatively non-specifically with the receptor, and that induction is greatly facilitated by the N-terminal domain that recognizes specifically its cognate receptor. The possibility that these peptide pheromones interact with the receptor via the membrane environment is in line with our hypothesis. Changes throughout the nisin molecule affect the signaling capacity, the A ring being essential (Dodd et al. 1996). It has been suggested that when nisin is inserted in the membrane the C-terminal part of the protein would not be inside the membrane and that the positive charge(s) would interact with the negatively charged phospholipids (van den Hooven et al. 1996). Additionally, charge alterations in the C-terminal part of nisin also resulted in a reduction of induction capacity (van Kraaij et al. 1997). Kuipers et al. (2007) reported that the C-terminal truncation of residues 23 to 34 reduced the induction capacity. Taking all the available data together, we suggest that it is the C-terminus of nisin that is available for NisK. The part of nisin needed for interaction with NisK may possibly stick out from the membrane into the aqueous pseudoperiplasmic space and be available for signal recognition by NisK (Fig 6). This part of nisin could well be involved in signaling function of nisin explaining why functional nisin inside the cells is capable of inducing the PnisZ promoter in the cells of LAC85. This could represent a general feature of amphiphilic peptides as signaling molecules in TCST pathways. The possibility that nisin interacts with NisK via the membrane environment is in line with the suggestion of van Belkum et al. (2007). 7.1.5. Isolation of natural mutants of the GFPuv nisin bioassay indicator L. lactis LAC275 cells by FACS(II) In this study we aimed to test the utilization of FACS to isolate natural mutants, with increased sensitivity to nisin induction, of a nisin-sensing L. lactis LAC275 indicator GFPuv bioassay strain. No mutagenesis procedure was included prior sorting to avoid the risk of creating multiple mutations. Prior to the sorting analysis, we induced the cell with 10 pg nisin ml-1. Approximately 10,000 cells/particles were sorted. The constructed fluorescence histograms revealed a population of fluorescing cells showing higher fluorescence intensities than the majority of the nisin induced L. lactis LAC275 cells. The sorted cells were plated on two M17G plates (1/10 and 1/20 of the sorted cells) and grown over night at 30 C°. The viability of L. lactis LAC275 cells was high as the majority (>90%) of the sorted cells survived the sorting 33


conditions compared to studies of Hakkila (2006) in which only 1–10% of the Escherichia coli cells survived in a similar sorting. Probably, the thick cell wall is giving rigidity to the L. lactis cells and affects viability in the sorting conditions. Sixty-eight pure cultures were isolated for further analysis. Sixty-six of these cultures showed a similar responsiveness to nisin as the parental LAC275 strain. Two mutant L. lactis LAC275 strains, LAC344 and LAC345, showed more sensitive linear responsiveness (0.2–0.7 pg ml-1) to nisin than the parental LAC275, which has a linear responsiveness in the range of 10–70 pg nisin ml-1. The sensitivity of LAC344 and LAC345 strains were in the same range of sensitivity achieved by Immonen and Karp (2007). In the low nisin concentrations (0.1–0.7 pg ml-1) the parental LAC275 cells were not expected to show any linear responsiveness to nisin (Hakovirta et al. 2006) as was evident as cells of this strain got induced first by nisin in concentrations above 10 pg ml-1. The obtained LAC344 and LAC345 strains in this study can be used as second generation assay strains of the GFPuv nisin bioassay for even more sensitive detection of nisin. Such strains are needed if the sample amount is scaled down for ‘‘lab-on-a-chip’’ applications. Such compact miniaturized devices allow samples to be analyzed at the point of need rather than in a centralized laboratory. While flow cytometry is a well-established tool for the characterization of eukaryotic cells and their interactions, it has apparently still not reached its full potential for microbiological applications (Steen 2000; Longbardi Givan 2001; Link et al. 2007). In our study, FACS was proven to be a useful method for rapid and accurate isolation of natural mutants having a desired phenotype.

7.2. Applied focus: screening of bacteriocinogenic lab strains for future feeding trials in chicken (III, IV) The genus Lactobacillus is essential for modern food and feed technologies. They are consumed by humans and fed to animals of commercial value as probiotics in efforts to maintain a balanced microbiota and to reduce the numbers of potential pathogens residing in the intestinal tract. Therefore, knowledge of the composition of chickens’ crop microbiota is critical for understanding the contribution of the microbiota members to the well-being of the avian host and for the selection of direct-fed microbials (DFM) commonly referred to as probiotics ( Flint and Garner 2009). 7.2.1. Characterization of the crop microbiota (III) In this study the crop was chosen as source for future selection of candidate probiotic since it has been suggested that the crop microbiota acts as a bacterial inoculum for the remainder of the gut (Fuller 1977, Lu et al. 2003). In addition, ingested pathogens will first come to the crop after ingestion and thereby crop could be a first line of defense if it harbors antipathogenic strains. Therefore, bacteria from crops of 1- and 5-week-old broiler chickens fed with two wheat-based diets (A and B) were grown on Lactobacillus-selective medium and identified (n = 300) based on partial 16S rRNA gene sequence. The most abundant Lactobacillus species were L. reuteri (33%), L. crispatus (18.7%), and L. salivarius (13.3%) (I, Table 1). L. reuteri was the most abundant species (P < 0.005) in the crops of the younger 34


chickens and significantly reduced in the crops of the 5-week-old chickens regardless of the feed (P = 0.016). The diversity of L. reuteri isolates was revealed by fatty acid analysis, whereas 94 L. reuteri isolates arranged into several clusters. Nisin has potential as a growth-promoting antibiotic (GPA). Therefore, we tested the nisin sensitivity of the strains of the most dominant bacteria, e. g. L. reuteri. If nisin would be used as a GPA it would be preferable if the strains of the most dominant specie would be resistant to nisin. Most of the sensitive isolates were found in younger chickens (77%) compared to the 5-week-old chickens (23%), whereas chickens fed with commercial feed B had a higher proportion of nisin-resistant isolates (73%) than did chickens fed with feed A (45%). The diversity of the L. reuteri population and the frequency of nisin resistant L. reuteri strains in the crops suggest that it could be possible to use nisin as a GPA without wiping out the dominant L. reuteri strains from the crop. Using nisin as a GPA could be combined with several strains representing different clusters and nisin resistance phenotypes in candidate feed supplements for chickens. 7.2.2. Characterization of L. salivarius bacteriocin which exhibit antagonistic activity against Campylobacter jejni (IV) The majority of LAB of animal microbiota is believed to be beneficial to the host. This study aimed at screening lactobacilli from chicken microbiota for antimicrobial activity against C. jejuni. Secondly, any strains exhibiting antibacterial activity were to be isolated and characterized for usage evaluation as bacteria potentially promoting chicken health or as potent anti-food-borne pathogens. From our collection (N = 40) of crop Lactobacillus salivarius many strains produced antibacterial activity against C. jejuni but LAB47 showed the strongest activity and the broadest activity range. Therefore, it was chosen for further studies including partial purification of the inhibitory substance. The bacteriocin was partially purified using ammonium sulphate precipitation and further purified by solid phase extraction. The isolated peptides were run on SDS-PAGE and the obtained polypeptides were overlaid with a bacteriocin sensitive Campylobacter culture identifying one of the polypeptides to have anti-camplylobacter activity. This polypeptide named salivaricin 47 was subjected to molecular mass analysis showing it to have a molecular mass of approximately 3.2 kDa, representing a novel size for bacteriocins of L. salivarius. The anti-Campylobacter activity of salivaricin 47 was not affected by catalase, but was abolished by the proteolytic enzymes, trypsin and Proteinase K. The salivaricin 47 was heat stable (15 min at 90°C) and showed inhibitory activity over a wide pH range (3 to 8). To our knowledge, only four bacteriocins from L. salivarius isolates have been purified and their primary structure has been characterized (Flynn et al. 2002; Stern et al. 2006; Busarcevic et al. 2008; Pingitore et al. 2009). Salivaricin 47 characterized in this study is a novel bacteriocin judged from the approximated molecular mass (about 3.2 kDa) and may be used for improving food safety regarding the threat caused by Campylobacter jejuni as well as be used as a protective culture in the crop of chickens potentially lowering the prevalence of Campylobacter in chickens. To date, four bacteriocins have been previously characterized and showed to have potent activity against C. jejuni (Table 5).

35


Table 5. Bacteriocins produced by Lactobacillus salivarius and bacteriocins with anti-C. jejuni activity. Bacteriocin-producer

Name of bacteriocin

ABP-118 OR-7 LS1 Salivaricin CRL 1328, Salα and Salβ Salivaricin47 L. salivarius Paenibacillus polymyxa SRCAM 602 E-760 Enterococcus durans Enterococcus faecium E 50–52 Lactobacillus salivarius L. salivarius L. salivarius L. salivarius

ND= not determined

36

Molecular mass kDa 4.09 5.13 10 4.096 and 4.33

Anticampylobacter activity ND + ND ND

References

3.20 3.86 5.36 3.34

+ + + +

This study Stern et al. 2005 Line et al. 2008 Svetoch et al. 2008

Flynn et al. 2002 Stern et al. 2006 Busarcevic et al. 2008 Vera Pingitore et al. 2009

Conclusions

8. CONCLUSIONS The induction of the PnisZ in LAC85 cells occurred most likely via NisK. The mechanism of how nisin reaches NisK from the cytoplasm is unclear, but it is possible that nisin inserts into the membrane and from there presents segments of the molecule into the pseudoperiplasmic space for the NisK signal recognition domain. This also suggests that in the wildtype nisin producers, NisK recognizes nisin not from the water-soluble phase but from the membrane. This may represent a general feature of amphiphilic peptides functioning as self-signaling molecules in TCST pathways as has also been suggested by van Belkum et al. (2007). FACS could easily be used to isolate natural mutants of L. lactis LAC275 without any mutagenesis, suggesting that the sensitivity of many other bioassays based on GFPuv induced production, may be easily improved in a similar manner. The obtained LAC344 and LAC345 strains can be used as second generation assay strains in the GFPuv nisin bioassay for even more sensitive detection of nisin. In this study, the most abundant Lactobacillus species in the 1- and 5-week-old chicken crops was L. reuteri. The diversity of the L. reuteri population suggests a need of including several strains representing different clusters and nisin resistance phenotypes in candidate probiotic feed supplements for chickens. The change in the nisin resistance of L. reuteri isolates with age suggests that adding nisin to feed as a potential growth promoter may negatively affect the L. reuteri population of young chickens, whereas this may be less influencial in older chickens. On the other hand, the nisin-resistant L. reuteri strains isolated in this study could be added to nisin-containing feed fed to young chickens, thereby potentially maintaining L. reuteri as part of the dominating crop microbiota. The nisin-sensitive L. reuteri isolates could be used as hosts for the transformation of plasmids using foodgrade nisin selection in attempts to add extra value or functions to the bacteria in the feed supplement. However, such strains would be defined as GMO in EU restricting their use to countries with less strict legislation. Feed and food safety is an important component of any new animal antimicrobial applications. The food safety problem, Campylobacter –contaminated broilers is a significant risk factor of human campylobacteriosis and hence the control of C. jejuni in poultry would reduce the risk of human exposure to Campylobacter. The chicken intestinal bacteriocin–producing Lactobacillus salivarius are believed to function as an innate barrier against pathogens in the GIT. Salivaricin 47 is a potent bacteriocin against C. jejuni and has potential use as a protective culture in the crop of chickens which may lower the prevalence of Campylobacter in chickens.

37

Future prospects

9. FUTURE PROSPECTS In this study the exact location of nisin as inducer molecule was not shown. A future goal is to determine if the activation of NisK by nisin in LAC85 is from nisin inserted in the membrane, nisin on the membrane surface or from soluble nisin in the pseudoperiplasmic space. One way to study this would be to use different mutants expressing nisin instead of the wild-type nisin and measure changes in nisin induction in relation to differences in hydrophilicity of the expressed nisin mutants. The mutant strains LAC344 and LAC345 (II) are natural mutants affecting some unknown factor leading to improved nisin sensitivity when the strains are used in the nisin GFPuv bioassay. One future prospect is to identify the mutations and analyze the mechanism by which these mutations affect the nisin sensitivity of the bioassay strains. The mutations may be isolated by cloning fragments from LAC85 into LAC275 and screen for effects on nisin induced fluorescence. The results in study III open up the possibility for feed studies adding nisin to the feed together with nisin resistant strains. A good CE preparation may require several strains and species. The three most common species in the chicken crop were L. reuteri, L. crispatus and L. salivarius, but nisin resistant strains were only screened among the L. reuteri isolates (III). Therefore, nisin resistant strains need to be identified among the L. crispatus and L. salivarius strains. Then, the feeding study with nisin and nisin resistant strains can begin. The anti-campylobacter activity of the chicken crop strain L. salivarius 47 urges further characterization. The isolated bacteriocin needs to be purified in higher quantities for detailed characterization including N-terminal amino acid sequencing, mass analysis, specific activity and spectrum of activity. A future prospect is also to clone the corresponding gene and eventual specific genes involved in biosynthesis of salvaricin 47. All such data would help to evaluate the potential of this bacteriocin in applications aiming at using the producer strain or the bacteriocin to improve food safety.

38

Acknowledgements

10. ACKNOWLEDGEMENTS The work described in this thesis was carried out at the Division of Microbiology, Department of Food and Environmental Sciences, University of Helsinki. I wish to express my deep gratitude to the each of the following: Centre of Excellence “Microbial Resources Research Unit” of Academy of Finland and the Research Foundation of University of Helsinki for personal grants, Rasio Ltd., and The Finnish Cultural Foundation for funding this thesis. Professor Per Saris for his thorough supervision, expert guidance, encouraging and inspiring discussions at all stages of my study. The pre-examiners professors Seppo Salminen and Matti Karp for carefully reviewing this thesis and giving me invaluable suggestions for improving it. Co-authors Juha Apajalahti, Kaisa Hakkila, Anu Surakka, Kari Kylä-Nikkilä, and Runar Ra for their valuable contribution to the papers. The present and former heads of our division, professors Per Saris, and Mirja SalkinojaSalonen, for providing excellent facilities for the research. Professor Kristiina Lindström for organizing excellent courses. Dr.Timo Takala for critical reading an earlier draft of the thesis and his invaluable comments. His scientific knowledge has great impact on our research group and is highly acknowledged. The present and former LAB group members: Janetta Hakovirta, Timo Takala, Päivi Ramu, Titta Manninen, Fang Cheng, Justus Reunanen, Sahar Navidghasemizad, Ulla Saarela, Ömer Simsek, Kari Kylä-Nikkilä, Ossian Saris, Yurui Tang, Ruiqing Li, and Adewale Ariwo-Ola, for creating a pleasant working atmosphere and for many relaxing food breaks. Janetta is warmly thanked for being a true friend over these years, for sharing thoughts and talks that could have been endless, and for the weekly jogging sessions we had together. My colleagues from the MSS research group, Camelia Constantin, Minna Peltola, Mari Raulio, Douwe Hoornstra, and Jaakko Ekman for their friendly attitudes and generous assistance with all manner of things. Irina Tsitko for her excellent help with FAME chromatography and for being such a cheerful person. All former and current colleagues and staff at the Division of Microbiology, especially David ,Taina, Pauliina, Aila, Marja, Beata, Lara, Outi, Pekka, Mannu, Riitta, Mika, Taru,Tarja, and Tuula for creating relaxed working atmosphere. Leena Steininger, Hannele Tukiainen and Tuula Suortti for all the secretarial help.

39

Acknowledgements

Dr. Ulrike Lyhs for the Campylobacter strains and introduction to the Campylobacter world. Terttu Terho from Turku Centre for Biotechnology for his brilliance with FACSVantage and cell sorting. Viikki Science Library for the excellent information service. I have been blessed with true friends: Mike, Manu, Ramzi, Fatima, Mona and Marah for sharing joys and sorrows. My heartfelt thanks go to my father and my mother for their everlasting love and support in all aspects of my life. My sister Taim and my brothers Ziyad and Kahled whom I missed so much during all this time and not forgetting their children, relatives, and my extended family. This dissertation is also dedicated to the memory of my father in law. The biggest love goes to my husband, Haki, for his love and years of standing by me, and to our son, Alharith and our daughter Dania “DoDo”. Your love, encouragement and patience have been vital to me and helped me finish this thesis. Helsinki, June 2010

Hanan Hilmi

40

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