凝结芽胞杆菌抗菌作用机制

凝结芽胞杆菌已经被广泛应用于功能性食品或食品添加剂,也已被应用于饲料添加剂。我国国家食品药品监督管理局在2005年批准青岛东海药业有限公司研制的凝结芽胞杆菌TBC-169菌株作为治疗肠道疾病的新药。关于凝结芽胞杆菌的功能和特性、培养及其生产方法等方面有大量文献。该文仅对凝结芽胞杆菌产生的凝固素的分子结构及其抗菌的作用机制作简单综述。     

一株产淀粉酶芽胞杆菌SY200的鉴定及其对动物病原菌的生物拮抗试验

目的对一株产淀粉酶芽胞杆菌SY200进行鉴定及其对动物病原菌的生物拮抗试验。方法提取芽胞杆菌SY200基因组DNA,采用细菌16S rRNA通用引物进行PCR扩增及对扩增到的目标片段的测序,将测序结果与NCBI上已知菌种的16S rRNA序列进行BLAST对比,并构建系统进化树进行分析。采用滤纸片法和牛津杯法分别研究该芽胞杆菌的全菌液及培养物上清液对3株病原菌的生物拮抗。结果结合细菌形态观察及生理生化特性鉴定,最终确定菌株SY200为甲基营养型芽胞杆菌(Bacillus methylotrophicus);芽胞杆菌SY200全菌培养液和培养上清液对产肠毒素大肠埃希菌、鸡白痢沙门菌、金黄色葡萄球菌均有较强的生物拮抗作用,抑菌物质主要为细菌的代谢产物。结论芽胞杆菌SY200被鉴定为甲基营养型芽胞杆菌,该菌株对3株动物性病原菌具有较强的生物拮抗作用。     

水产源芽胞杆菌的分离鉴定及生物学功能分析

筛选具有较好生物学功能的芽胞杆菌(Bacillus),用以改善池塘养殖过程饲料等有机物降解、抑制水体中的病原菌,对从健康养殖鱼虾塘水体中分离的菌株,进行生化试验和16S rDNA序列分子鉴定;通过产酶、耐酸、耐高温试验,抑菌试验以及安全性试验研究分离菌株的生物学功能。试验共筛选出8株细菌,经生化试验及16S rDNA序列的分子鉴定,确定菌株ZHX17、ZHX18、ZHX31为贝莱斯芽胞杆菌（Bacillus velezensis）,菌株ZHX14、ZHX15为地衣芽胞杆菌（Bacillus licheniformis）、菌株NSX4、NSX7、NSX9为枯草芽胞杆菌（Bacillus subtilis）。试验结果表明,8株芽胞杆菌均具有较强的耐酸、耐高温特性,其中3株贝莱斯芽胞杆菌具有较强的分泌淀粉酶、纤维素酶、蛋白酶的能力,抑菌效果好于其他5株芽胞杆菌。安全性试验结果表明,8株芽胞杆菌对草鱼、罗非鱼相对安全。8株芽胞杆菌同时具备分泌淀粉酶、纤维素酶和蛋白酶3种胞外酶的能力,其中贝莱斯芽胞杆菌具有较强的病原菌抑菌能力,可以作为病原拮抗益生菌做进一步研究。     

海洋中分离的抗白念珠菌芽胞杆菌性状特征及系统鉴定

对筛选自中国南海、黄海、渤海4个地区近海海水样品的7株有抗白念珠菌活性,且稳定性较强的芽胞杆菌的形态特征、培养特征及生理生化试验等进行系统分析比较,结果表明,LU-B02为凝结芽胞杆菌(Bacillus coagulans),LU-B13为蜡样芽胞杆菌(Bacillus cereus),其他各菌株为短小芽胞杆菌(Bacillius pumilus),除LU-B02耐盐度5%外,其他耐盐度达10%以上。它们均属于芽胞杆菌的第一群。16S r DNA基因同源性序列比较进一步证实LU-B02为凝结芽胞杆菌,它与凝结芽胞杆菌标准菌株ATCC15950在鉴定特征上虽然相同,但与后者相比,最高生长温度较低,耐盐性较强,可以利用阿拉伯糖、木糖、甘露醇等碳源,可水解酪素,并可稳定地产生抗白念珠菌活性物质,将其命名为Bacillus coagulans subsp.heishijiaosis。尚未见抗白念珠菌的凝结芽胞杆菌菌株的有关报道。     

产乳酸凝结芽胞杆菌N001的抗逆性研究

目的研究前期经初筛的产乳酸凝结芽胞杆菌N001芽胞的抗逆性。方法在模拟饲料制粒条件下和动物消化道内逆境条件下的存活能力,测定N001芽胞的抗热、抗酸、耐胆盐性能和对抗生素的敏感性。结果凝结芽胞杆菌N001芽胞具有很强的耐高温、耐酸、耐胆盐能力;同时N001芽胞对营养体敏感的抗生素也有良好的耐受性。结论凝结芽胞杆菌N001芽胞具有很强的抗逆性,可以作为益生菌制剂的良好菌种。     

野生东亚钳蝎消化道可培养细菌的分离鉴定、耐药性及产消化酶活性

目的分离东亚钳蝎消化道中的可培养细菌,并研究其产消化酶活性和耐药性,探讨消化道细菌对东亚钳蝎消化食物的影响。方法野生东亚钳蝎采用传统细菌分离培养方法分离其消化道内可培养细菌,利用16S rRNA序列进行细菌的分子鉴定;利用平板透明圈法测定可培养细菌产淀粉酶、蛋白酶、脂肪酶和纤维素酶等消化酶的活性;采用药敏纸片扩散法进行消化道可培养细菌的药敏试验。结果在东亚钳蝎消化道中共分离到4个属10种细菌,其中芽胞杆菌属7种,分别为蛋白水解芽胞杆菌、解淀粉芽胞杆菌、巨大芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌、婴儿芽胞杆菌和长形赖氨酸芽胞杆菌;葡萄球菌属、纤维微菌属和短波单胞菌属各1株,分别为松鼠葡萄球菌、纤维化纤维微细菌和泡囊短波单胞菌。对10种细菌的产消化酶活性分析表明,8种细菌有产消化酶活性,占细菌总数的80%;5种细菌有产蛋白酶活性,分别为蛋白水解芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌、松鼠葡萄球菌和纤维化纤维微细菌;7种细菌有产淀粉酶活性,分别为蛋白水解芽胞杆菌、解淀粉芽胞杆菌、巨大芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌、松鼠葡萄球菌和婴儿芽胞杆菌;未筛选到产纤维素酶和脂肪酶活性的细菌;蛋白水解芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌和松鼠葡萄球菌可同时产2种消化酶。在药敏试验中,8种细菌对多粘菌素B耐药,5种细菌对四环素耐药,3种细菌对万古霉素耐药。结论东亚钳蝎消化道中可培养细菌的组成相对单一,多数细菌有分泌淀粉酶和蛋白酶的功能,说明东亚钳蝎消化道中的细菌可能有协助其进行食物消化的功能。消化道细菌对抗生素多表现为敏感,推测其生存环境中抗生素的污染较少,同时在对东亚钳蝎人工饲养的场所进行消毒时,要谨慎选择抗生素,防止抗生素使用不当致使东亚钳蝎肠道菌群紊乱引起疾病。     

一株凝结芽胞杆菌的筛选及对肉鸡生产性能的影响

目的分离自然界中可安全应用于畜禽的凝结芽胞杆菌,提高肉鸡的饲料消化吸收、降低料肉比,改善生产性能。方法在山上、海边及樱桃树下的土壤中分离凝结芽胞杆菌,通过生理生化、16S rRNA鉴定研究菌株的生物学特性,并考察其安全性;最终应用于促进饲料消化吸收、保护肠道健康、提高生产性能。肉鸡饲养至第31天时开始添加凝结芽胞杆菌10 d,分为实验组(1、2、3组)和对照组,考察生产性能指标确定其功能。结果凝结芽胞杆菌J-1产酸溶钙圈高于其他2株,为革兰阳性菌。实验表明在同样的养殖条件下,实验组鸡的粪便干燥、成型、无酱黄色出现;实验组鸡苗的前期7日龄时体质量低于对照组,肉鸡的成活率分别高于对照组0.9%、0.7%、1.2%,料肉比分别低于对照组0.066、0.080、0.050,出栏均重分别优于对照组0.144 kg、0.129 kg、0.079 kg。结论筛选出1株安全、产酸且应用肉鸡实际效果优良的凝结芽胞杆菌,可降低肉鸡料肉比,提高肉鸡的出栏均重、成活率、饲料利用率,并减少肉鸡养殖后期抗生素的使用。     

辣椒镰孢根腐病防病促生细菌的筛选及其效应

【目的】筛选辣椒(Capsicum annuum L.)根腐病防病促生细菌并明确其防病促生效应。【方法】采集健康辣椒根围土壤样品,以辣椒根腐病病原真菌茄镰孢(Fusarium solani)和尖镰孢(Fusarium oxysporum)为指示菌,采用平板对峙法筛选生防细菌,采用选择性培养基筛选溶无机磷、溶有机磷、固氮菌和解钾菌等促生菌,钼锑抗比色法测定溶磷量,凯氏定氮法测定固氮量,火焰原子吸收光谱法测定解钾量。对特性良好组合的菌株进行16S rDNA序列分析鉴定并制作菌剂,最后采用盆栽法测定菌剂防病促生效果。【结果】共筛选得到323株特性良好的功能菌株,拮抗菌78株,溶有机磷菌87株,溶无机磷菌107株,固氮菌128株,解钾菌123株,部分菌株同时具有多个功能特性。互作组合得到6个特性良好的菌株组合,包括8株功能菌株,鉴定发现XP271和XP181为枯草芽胞杆菌(Bacillus subtilis),XP125为特基拉芽胞杆菌(Bacillus tequilensis),XP236为耐盐芽胞杆菌(Bacillus halotolerans),XP79为巨大芽胞杆菌(Bacillus ...     

1株产琼脂酶细菌的分离鉴定及其胞外酶活性测定

从四川省成都市青城山采集土壤,以琼脂作为唯一碳源,筛选到产琼脂酶细菌CMCK136;通过形态观察、生化鉴定、16S r DNA测序及序列分析鉴定其种属;随后测定了菌株CMCK136的胞外酶活性。菌株CMCK136被鉴定为芽胞杆菌属细菌,命名为Bacillus sp.CMCK136。菌株CMCK136的胞外琼脂酶的最适酸碱度为p H 7.0,最适温度为35℃。菌株CMCK136是产琼脂酶细菌家族的新成员,该菌株的发现进一步提示芽胞杆菌属很可能蕴含有尚待开发的琼脂酶资源。     

产酶芽胞杆菌的筛选及菌株HB13000的鉴定

芽胞杆菌产酶种类多、生长速度快、抗逆性强,在堆肥过程中发挥着重要作用。本研究采用水解圈法研究了348株芽胞杆菌产酶特性,获得产纤维素酶、蛋白酶、淀粉酶芽胞杆菌34株。其中,菌株HB13000相对酶活力较高,酶活Hc(水解圈直径/菌落直径)分别为:纤维素酶Hc=4.30,蛋白酶Hc=1.47,淀粉酶Hc=3.25,对纤维素、淀粉和蛋白质均表现出较强的降解活性。依据菌体形态、生理生化特征和16S r DNA系统发育分析,鉴定HB13000为Bacillus methylotrophicus(甲基营养型芽胞杆菌)。本研究为有机物降解与农业废弃物堆肥提供了新的菌种资源。     

Concomitant Synthesis of Bacteriocin and Bacteriocin Inactivator from Serratia marcescens

We have found that Serratia marcescens strain P & S is bacteriocinogenic. However, the phenotypic expression of bacteriocin activity depends upon the temperature at which the cells are grown. When the organism is grown at 30 to 37 C, no bacteriocin activity can be demonstrated, whereas when it is grown at 39 C bacteriocin activity is readily observed. It appears that the P & S strain concomitantly synthesizes a bacteriocin and a substance which not only can inactivate the bacteriocin but also has a high activation energy for inactivation. This inactivator readily loses its activity when heated at 39 C for 1 hr. Two mutants were isolated from the P & S strain which can produce active bacteriocin when grown at temperatures from 30 to 39 C. It is significant that these mutants have considerably less bacteriocin inactivator. The data suggest that the inactivator is an extracellular protease. The ability of one of these mutants, JF58-12, to produce active bacteriocin at temperatures between 30 and 39 C is a stable property, whereas in the other mutant, JF48W, this property is unstable. JF48W was selected from the P & S strain in two steps: first a streptomycin-resistant variant (strain A-10) was isolated and from this mutant a strain (JF48W) was isolated which not only synthesized little of the inactivator but also did not synthesize the red pigmnet prodigiosin. This latter pleiotropic mutant appears to revert in one step to a phenotype similar to the P & S strain, since it is streptomycin-sensitive and produces prodigiosin and normal amounts of inactivator and the demonstration of bacteriocin activity is temperature-dependent.     

Identification and characterization of leucocyclicin Q, a novel cyclic bacteriocin produced by Leuconostoc mesenteroides TK41401

The culture supernatant of Leuconostoc mesenteroides TK41401, isolated from Japanese pickles, possessed antimicrobial activity against broad range of a bacterial genera and particularly strong activity against Bacillus coagulans, the major contaminant of pickles. An antimicrobial peptide was purified in three chromatographic steps, and its molecular mass was determined to be 6,115.59 Da by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). The primary structure of this peptide was determined by amino acid and DNA sequencing, and these analyses revealed that it was translated as a 63-residue precursor. This precursor showed high similarity to the precursor of lactocyclicin Q, a cyclic bacteriocin produced by Lactococcus sp. strain QU 12. The molecular weight calculated after cyclization, which was presumed to involve the same process as in lactocyclicin Q (between L3 and W63), agreed with that estimated by ESI-TOF MS. This peptide was proved to be a novel cyclic bacteriocin, and it was termed leucocyclicin Q. The antimicrobial spectrum of this bacteriocin clearly differed from that of lactocyclicin Q, even though their primary structures were quite similar. This is the first report of a cyclic bacteriocin produced by a strain of the genus Leuconostoc.     

Characterization of a bacteriocin produced by Enterococcus faecium GM-1 isolated from an infant

AIM: To partially characterize the bacteriocin produced by the GM-1 strain of Enterococcus faecium, isolated from the faeces of a newborn human infant. METHODS AND RESULTS: The bacteriocin produced by E. faecium GM-1 showed a broad spectrum of activity against indicator strains of Escherichia coli, Staphylococcus aureus, Vibrio spp., Salmonella typhimurium, Listeria monocytogenes, Lactobacillus acidophilus, and Streptococcus thermophilus. Treatment of the GM-1 bacteriocin with proteolytic enzymes reduced its inhibitory activities. The bacteriocin was stable at 100 degrees C for 20 min and displayed inhibitory activity at neutral pH. The optimal production of bacteriocin from E. faecium GM-1 was obtained when the culture conditions were pH 6.0-6.5 and 35-40 degrees C. The inhibitory activity of the bacteriocin was not substantially changed by the use of different carbon sources in the media, except when galactose was substituted for glucose. The use of a sole nitrogen source caused a decrease in inhibitory activity. A bacteriocin gene similar to enterocin P was identified from the total DNA of E. faecium GM-1 by PCR and direct sequencing methods. CONCLUSION: E. faecium GM-1, which was isolated from the faeces of a newborn baby, produces an enterocin P-like bacteriocin with inhibitory activity against Gram-positive and Gram-negative bacteria, including food-borne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium GM-1, isolated from infant faeces, produces a new bacteriocin that is similar to enterocin P. This bacteriocin is heat stable and has a broad antibacterial spectrum that includes both Gram-positive and Gram-negative bacteria.     

Nisin Production by Enterococcus hirae DF105Mi Isolated from Brazilian Goat Milk

The purpose of this study was to select the promising biopreservation bacteriocin producer strain from goat milk and characterize the expressed bacteriocin, related to its physiological and biochemical properties and specificity of operon encoding production and expression of antimicrobial peptide. Brazilian goat milk was used as the source for the selection of bacteriocin-producing lactic acid bacteria. One strain (DF105Mi) stood out for its strong activity against several Listeria monocytogenes strains. Selected strain was identified based on the biochemical and physiological characteristics and 16s rRNA analysis. The bacteriocin production and inhibitory spectrum of strain DF105Mi were studied, together with the evaluation of the effect of temperature, pH, and chemicals on bacteriocin stability and production, activity, and adsorption to target cells as well as to the cell surface of bacteriocin producers. Physiological and bio-molecular analyses based on targeting of different genes, parts of nisin operon were performed in order to investigate the hypothesis that the studied strain can produce and express nisin. Based on biochemical, physiological, and 16s rRNA analysis, the strain DF105Mi was classified as Enterococcus hirae. The selected strain produces a bacteriocin which is stable in a wide range of pH (2.0–12.0), temperature (up to 120 °C), presence of selected chemicals and presents adsorption affinity to different test organisms, process influenced by environmental conditions. Higher bacteriocin production by Ent. hirae DF105Mi was recorded during stationary growth phase, but only when the strain was cultured at 37 °C. The strain’s genetic analysis indicated presence of the genes coding for the production of the bacteriocin nisin. This result was confirmed by cross-checking the sensitivity of the produced strain to commercial nisin A. The strong anti-Listeria activity, bacteriocin adsorption, and stability of produced bacteriocin indicate that Ent. hirae DF105Mi presents a differentiated potential application for biopreservation of fermented dairy products.

     

Comparative effectiveness of nisin and bacteriocin J46 at different pH values

E. HUOT, C. BARRENA-GONZALEZ AND H. PETITDEMANGE. 1996. A Comparative study of the inhibitory activity of nisin, the well-known lantibiotic produced by certain strains of Lactococcus lactis subsp. lactis , and of the bacteriocin produced by L. lactis subsp. cremoris J46, a strain previously isolated from fermented milk, was conducted. For both bacteriocins, the activity against L. lactis subsp. cremoris decreased with increasing pH. In addition, the bacteriocin preparations were more stable at 4 than at 20°C. The influence of the storage temperature was more crucial for nisin. Essentially the same activity was observed for bacteriocin J46 stored for 3 h at 4 or 20°C. More interesting was the observed stability of bacteriocin J46 at pH values between 5.8 and 6.8. For example, about 23% of nisin activity was lost at pH 6.4 whereas no loss of bacteriocin J46 activity was observed.     

Sequence analysis of the plasmid pColG from the Escherichia coli strain CA46

The complete 4715 nucleotide sequence of the plasmid pColG from the Escherichia coli strain CA46, which was originally assumed to code for colicin G activity, has been determined. Based on the nucleotide sequence homology of the 1828bp replication region, with an average G+C content of 48%, pColG was classified as a ColE1-like plasmid. Computer assisted analysis of the remaining 2887bp nucleotide sequence with an average G+C content of 34% revealed three putative OFRs. To find out whether one or all of the three ORFs code for a possible bacteriocin, a DNA fragment encompassing these ORFs has been cloned and the recombinant colonies tested for bacteriocin production. None of the colonies had an inhibitory activity against E. coli strains DH5, HB101 and MC4100. The assumption that the plasmid pColG from the E. coli strain CA46 codes for a bacteriocin thus could not be confirmed.     

Properties of the strains<Emphasis Type="Italic">Enterococcus haemoperoxidus</Emphasis> and<Emphasis Type="Italic">E. moraviensis</Emphasis>, new species among enterococci

Antibiotic susceptibility or resistance, urease activity, detection of the structural genes for bacteriocin production, bacteriocin activity as well as sensitivity of the isolates to enterocins (Ent) A and M were determined in 23 isolates of new species Enterococcus haemoperoxidus and E. moraviensis. The majority of the strains were antibiotic sensitive and exhibited low urease activity (< 10 nkat/mL). The most frequently detected genes for Ent were entA and entP. However, only the strain 466 of E. haemoperoxidus produced an antibacterial substance with inhibitory activity against 21 G+ indicators. It was partially purified reaching an activity of up to 12 800 AU/mL. This bacteriocin active strain also possessed the genes for EntA and EntP. The other strains did not inhibit the indicator strains. The substance produced by the 466 strain was active even after a 5-months storage at +4 and -20 degrees C. This substance has proteolytic and hydrophilic character, pH optimum of bacteriocin production by this strain being between 4 and 7. While E. moraviensis strains showed sensitivity to EntA (produced by E. faecium EK13) and to EntM (produced by E. faecium AL41), E. haemoperoxidus strains were sensitive to EntA (except strain 382) but less sensitive to the treatment by EntM.     

Isolation and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi

Aims:  Screening and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi, a traditional Korean fermented vegetable.
Methods and Results:  A total of 1000 lactic acid bacteria were isolated from various Kimchi samples and screened for the production of bacteriocin. Pediocin K23-2, a bacteriocin produced by the Pediococcus pentosaceus K23-2 strain, showed strong inhibitory activity against Listeria monocytogenes . The bacteriocin activity remained unchanged after 15 min of heat treatment at 121°C or exposure to organic solvents; however, it diminished after treatment with proteolytic enzymes. The bacteriocin was maximally produced at 37°C, when the pH of the culture broth was maintained at 5·0 during the fermentation, although the optimum pH for growth was 7·0. The molecular weight of the bacteriocin was about 5 kDa according to a tricine SDS-PAGE analysis.
Conclusions:  Pediococcus pentosaceus K23-2 isolated from Kimchi produces a bacteriocin, which shares similar characteristics to the Class IIa bacteriocins. The bacteriocin is heat stable and shows wide antimicrobial activity against Gram-positive bacteria, especially L. monocytogenes .
Significance and Impact of the Study:  Pediocin K23-2 and pediocin K23-2-producing P. pentosaceus K23-2 could potentially be used in the food and feed industries as natural biopreservatives, and for probiotic application to humans or livestock.     

Characterization, N-terminal sequencing and classification of cerein MRX1, a novel bacteriocin purified from a newly isolated bacterium: Bacillus cereus MRX1

AIM: To purify and characterize the bacteriocin produced by strain MRX1. METHODS AND RESULTS: A bacteriocin-producing strain was isolated and identified as Bacillus cereus. The bacteriocin, called cerein MRX1, was purified from the culture supernatant using hydrophobic interaction, cation-exchange chromatography and RP-HPLC. It could also be purified in abundance from the cell surfaces of the producer strain. Mass spectrometry revealed its molecular mass of 3137.93 Da. Sequencing of chemically modified bacteriocin identified its partial sequence: DWTCWSCLVCAACSVELL. Amino acid analysis, confirmed by (1)H-NMR, suggested cerein MRX1 to be a class II bacteriocin. This bacteriocin was remarkably hydrophobic, heat-stable and could withstand a wide range of pH. It exhibited a bactericidal mode of action against Bacillus coagulans JCM 2257(T). Cerein MRX1 was especially active against spoilage bacteria such as Bacillus subtilis and Listeria innocua (MICs in the 1 microg ml(-1) range). In contrast, lactic acid bacteria were resistant or required higher concentrations to be inhibited. CONCLUSIONS: Cerein MRX1 is similar by its N-terminal sequence to thuricin 17 recently isolated from Bacillus thuringiensis NEB17. However, the two bacteriocins are different by their molecular masses and amino acid compositions. SIGNIFICANCE AND IMPACT OF THE STUDY: Chemical stability of cerein MRX1 and its ability to inhibit a large number of undesirable bacteria may give an advantage to its food or clinical application as an antibacterial agent.     

Isolation and purification of propionicin PLG-1, a bacteriocin produced by a strain of Propionibacterium thoenii.

Production of propionicin PLG-1 by Propionibacterium thoenii P127 was pH dependent, with maximal activity detected in supernatants of cultures grown at pH 7.0 Propionicin PLG-1 was purified by ion-exchange chromatography and isoelectric focusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of propionicin PLG-1 purified through isoelectric focusing resolved a protein band with a molecular weight of 10,000. Propionicin PLG-1 was bactericidal to sensitive cells, demonstrating single-hit kinetics. The producing strain harbored a single plasmid (pLG1) with an approximate size of 250 kb. Preliminary data indicate that both propionicin PLG-1 and immunity to the bacteriocin are encoded on the chromosome. Exposure of strain P127 to acriflavine or to N-methyl-N'-nitro-N-nitrosoguanidine yielded isolates that no longer produced bacteriocin activity and isolates that were cured of the plasmid. However, loss of bacteriocin production was not correlated with loss of the plasmid. Isolates cured of the plasmid were phenotypically identical to plasmid-bearing cells in fermentation patterns, pigment production, and growth characteristics.