Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

Rapid analysis of two food-borne microbial communities at the species level by Fourier-transform infrared microspectroscopy

The species composition of microbial communities in natural habitats may be extremely complex and therefore a quantitative analysis of the fraction each species contributes to the consortium has proven to be difficult. During recent years, the identification of bacterial pure cultures based on their infrared spectra has been established. Fourier-transform infrared microspectroscopy now proceeds a step further and allows identification of microorganisms directly plated from community dilutions. Infrared spectra of microcolonies of 70-250 microm in diameter can be recorded without producing a pure culture of the isolate. We have applied this novel technique for quantitative comparative analysis of two undefined, geographically separated food-borne smear cheese microbial consortia of limited complexity. Due to the high degree of automation, up to 200 microcolonies could be identified in 1 day and, in total, 3170 infrared spectra of microcolonies were recorded. The results obtained have been verified by Fourier-transform infrared macrospectroscopy and 16S rDNA sequencing. Interestingly, although the communities were unrelated, Staphylococcus equorum, Corynebacterium casei, Arthrobacter casei and Brevibacterium linens were found to be part of both consortia, however, with different incidence. In addition, Corynebacterium variabile, Microbacterium gubbeenense, Brachybacterium alimentarium, Enterococcus faecalis and an unknown species were detected in either one of the consortia.     

Quantitative detection of Corynebacterium casei in cheese by real-time PCR

The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10(5) to 10(10) CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 10(5) CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.     

Sources of the adventitious microflora of a smear-ripened cheese

AIMS: To determine the relationships between the major organisms from the cheese-making personnel and environment and the surface of a smear cheese. METHODS AND RESULTS: 360 yeast and 593 bacteria from the cheese surface, the dairy environment and the hands and arms of personnel were collected. Pulsed-field gel electrophoresis, repetitive sequence-based polymerase chain reaction and 16S rDNA sequencing were used for typing and identifying the bacteria, and mitochondrial DNA restriction fragment length polymorphism and Fourier-transform infrared spectroscopy for typing and identifying the yeast. The three most dominant bacteria were Corynebacterium casei, Corynebacterium variabile and Staphylococcus saprophyticus, which were divided into three, five and seven clusters, respectively, by macrorestriction analysis. The same clones from these organisms were isolated on the cheese surface, the dairy environment and the skin of the cheese personnel. Debaryomyces hansenii was the most dominant yeast. CONCLUSIONS: A 'house' microflora exists in the cheese plant. Although the original source of the micro-organisms was not identified, the brines were an important source of S. saprophyticus and D. hansenii and, additionally, the arms and hands of the workers the sources of C. casei and C. variabile. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the major contribution of the house microflora to the ripening of a smear-ripened cheese has been demonstrated.     

Survival of surface ripening cultures during storage and monitoring their development on cheese

AIMS: To study the survival of bacteria isolated from the surface of smear cheese and monitor their development during cheese ripening. METHODS AND RESULTS: The storage of five potential bacterial surface-ripening cheese cultures, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variable, Microbacterium gubbeenense and Staphylococcus saprophyticus, in maximum recovery diluent (MRD), containing 0.85% w/v or 5% w/v NaCl, at 21 or 4 degrees C for 40 days, was investigated. All five strains studied survived well with a maximum decrease of c. 2.5 log(10) CFU ml(-1) after storage for 40 days at 4 degrees C in 0.85% or 5% w/v NaCl. Survival, especially of C. variable, was less at 21 degrees C. The development of defined ripening cultures containing C. casei and Debaryomyces hansenii on two farmhouse cheeses was also evaluated. Using pulsed-field gel electrophoresis (PFGE) for the bacteria and mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) for the yeast, it was shown that the ripening cultures could be re-isolated in high numbers, 10(8) CFU cm(-2) for C. casei and 10(6) CFU cm(-2) for D. hansenii, from the cheese surface after 2.5 weeks of ripening. CONCLUSIONS: Ripening strains of surface ripening cultures can be stored in MRD containing 5% w/v salt at 4 degrees C for at least 40 days. Such cultures are recovered in high numbers from the cheese during ripening. SIGNIFICANCE AND IMPACT OF STUDY: This study has provided a low-cost and efficient way to store bacteria that could be used as ripening cultures for smear cheese. Such cultures can be recovered in high numbers from the cheese surface during ripening.     

Surface microflora of four smear-ripened cheeses

The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and Debaryomyces hansenii was the dominant species in the four cheeses. Yarrowia lipolytica was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species, Corynebacterium casei, Corynebacterium variabile, Arthrobacter arilaitensis, Arthrobacter sp., Microbacterium gubbeenense, Agrococcus sp. nov., Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus saprophyticus, Micrococcus luteus, Halomonas venusta, Vibrio sp., and Bacillus sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria, C. casei and A. arilaitensis, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses.     

Biodiversity of the bacterial flora on the surface of a smear cheese

The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with Brevibacterium linens BL2) and noninoculated (not deliberately inoculated with B. linens BL2) cheese were investigated. The results show that, contrary to accepted belief, B. linens is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative Staphylococcus spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of Corynebacterium casei (50.2% of isolates), Corynebacterium mooreparkense (26% of isolates), and Microbacterium gubbeenense (12.8% of isolates). In addition, five of the isolates from the inoculated cheese were Corynebacterium flavescens. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species. C. mooreparkense and C. casei grew at pH values below 4.9 in the presence of 8% NaCl, while M. gubbeenense did not grow below pH 5.8 in the presence of 5 to 10% NaCl. B. linens BL2 was not recovered from the inoculated cheese because it was inhibited by all the Staphylococcus isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.     

斜纹夜蛾幼虫肠道细菌分离鉴定及其功能初步分析

昆虫肠道微生物对其寄主的生长发育、营养代谢、免疫以及农药抗性等方面都发挥着重要作用。为研究斜纹夜蛾Spodoptera litura幼虫肠道细菌的多样性,并为其功能验证做准备,本文利用传统微生物分离纯培养方法从斜纹夜蛾4龄幼虫肠道中共分离鉴定得到10株细菌,分别为属于变形菌门(Proteobacteria)的脱氮假单胞菌(Pseudomonas denitrificans),不动细菌(Acinetobacter sp.),肺炎克雷伯氏菌(Klebsiella pneumoniae)和肠杆菌(Enterobacter sp.);属于厚壁菌门(Firmicutes)的鸡葡萄球菌(Staphylococcus gallinarum),蒙氏肠球菌(Enterococcus mundtii),蜡样芽胞杆菌(Bacillus cereus)和枯草芽胞杆菌(Bacillus subtilis)以及放线菌门(Actinobacteria)的微杆菌(Microbacteriums sp.)和乳酪棒杆菌(Corynebacterium casei)。变形菌门和厚壁菌门是斜纹夜蛾肠道可培养细菌中的优势菌群。功能验证实验表明肠杆菌具备纤维素降解能力,微杆菌具备很强的苯酚降解能力。本研究为未来深入研究斜纹夜蛾肠道微生物的功能提供了方向和菌株材料。     

Antibody for detection and quantitation of membrane-associated folate-binding protein from Lactobacillus casei

Lactobacillus casei cells contain a 25 kDa, membrane-associated, folate-binding protein (fbp), which is a component of the folate transport system. Polyclonal antibody to fbp (anti-fbp) has been prepared, and conditions have been established for detection and quantitation of the protein. Anti-fbp did not block [3H]folate transport or binding in L. casei cells. As judged by Western blots, the antibody reacted only with fbp on sodium dodecyl sulfate electrophoretograms of Triton X-100 extracts of L. casei membranes. Anti-fbp showed no cross-reactivity with L. casei dihydrofolate reductase, L. casei 5,10-methenyltetrahydrofolate synthetase, L1210 dihydrofolate reductase, rat liver dihydrofolate reductase, or L1210 folate-binding protein. Enzyme-linked immunosorbent assay measurements indicated the presence of an fbp in membranes of Lactobacillus salivarius and two transport-defective sublines of L. casei. Anti-fbp was used to demonstrate selective extraction, with n-butanol, of fbp from a mixture of Triton-solubilized L. casei membrane proteins; repression of fbp in membranes of L. casei cells grown on high levels of folate; and localization of fbp by electron microscopy, using anti-fbp in conjunction with goat anti-rabbit IgG gold conjugate, in L. casei membranes.     

表达鸡传染性法氏囊病毒VP2蛋白的干酪乳杆菌免疫保护效力

利用干酪乳杆菌作为传染性法氏囊病毒(IBDV)VP2抗原传递系统,探讨口服雏鸡的免疫次数、免疫剂量、免疫途径和攻毒保护效果。用pLA-VP2重组干酪乳杆菌对5日龄雏鸡进行二次和三次免疫,并设108、109、1010 CFU/mL的重组干酪乳杆菌组,间接ELISA检测血清IgG和小肠洗液sIgA,末免后7 d攻毒,计算保护效果。根据确定的2次免疫和109 CFU/mL免疫剂量免疫5日龄雏鸡,分别口服、滴鼻/点眼pLA-VP2/L.casei,口服、肌注商品活苗及口服pLA/L.casei和PBS为对照,监测IgG和sIgA抗体水平;末免后7 d检测脾淋巴细胞增殖情况并攻毒,7 d后剖检,观察法氏囊损伤程度并记录病变得分和保护率。结果表明各组的特异性sIgA、IgG抗体水平显著高于对照组(P0.01);口服pLA-VP2/L.casei组的淋巴细胞刺激指数显著高于其他组(P0.01),保护率高达83.3%,免疫保护效果优于滴鼻/点眼组。因此,构建的重组干酪乳杆菌的安全性优于商品活苗,可以作为IBDV候选疫苗。     

A new synbiotic, Lactobacillus casei subsp. casei together with dextran, reduces murine and human allergic reaction

We studied the development of atopic dermatitis-like skin lesions in NC/Nga mice and the allergic symptoms and blood patterns of healthy volunteers during the cedar (Cryptomeria japonica) pollen season in Japan following oral administration of a new synbiotic, Lactobacillus casei subsp. casei together with dextran. The combination of L. casei subsp. casei and dextran significantly decreased clinical skin severity scores and total immunoglobulin E levels in sera of NC/Nga mice that had developed picryl chloride-induced and Dermatophagoides pteronyssinus crude extract-swabbed atopic dermatitis-like skin lesions. During the most common Japanese cedar pollen season, synbiotic L. casei subsp. casei and dextran in humans led to no significant changes in total nasal and ocular symptom scores, in the levels of cedar pollen-specific immunoglobulin E, interferon-gamma and thymus and activation regulated chemokine or in the number of eosinophils in sera, whereas the placebo group showed a tendency for increased levels of cedar pollen-specific immunoglobulin E, thymus and activation regulated chemokine and number of eosinophils, and a decrease in interferon-gamma levels. Thus, the oral administration of synbiotic L. casei subsp. casei together with dextran appears to be an effective supplement for the prevention and treatment of allergic reactions.     

Lactose metabolism and lactase gene sequence homologies amongst lactobacilli

A number of strains of Lactobacillus spp., including the thermophilic and mesophilic dairy species, were screened for the presence of β -galactosidase ( β -gal) and phospho- β -galactosidase (pbg) enzyme activities. The majority of lactose fermenting strains exhibited β -gal rather than pbg enzyme activity with the highest levels in the thermophilic dairy species.
Correlation between these enzymes and the presence of specific genetic determinants was sought using probes for β -gal and pbg genes from Lactobacillus casei ssp. casei strain 64H. Southern transfer and filter hybridization showed that the β-gal probe shared homology with one strain of Lact. casei ssp. casei only. Sequences homologous to the pbg gene were detected only in plasmid DNA from the same strain of Lact. casei ssp. casei and with plasmid DNA from an apparently unrelated strain of Lactobacillus which exhibited no pbg activity. Two other strains of Lact. casei ssp. casei appeared to show homology between their chromosomal DNA and the pbg gene probe. No other homologies were detected. Therefore, although lactase activity could be detected in many strains of Lactobacillus spp., the genetic determinants involved did not share extensive homology.     

Detection of permanent Lactobacillus casei subsp. casei strains in weaned infants' gut

The Lactobacillus microflora in the faeces of two infants was followed in the days after weaning. All colonies isolated at the highest dilution were Lactobacillus casei subsp. casei . They were identified to strain level by plasmid profile, total soluble cell protein and surface cell protein pattern. Two dominant strains of L. casei subsp. casei were identified on successive days, which suggests that these were permanent members of the infant bowel microflora.     

Ribotyping ofLactobacillus casei group strains isolated from dairy products

A series of lactobacilli isolated from dairy products were characterized using biotyping and ribotyping with EcoRI and HindIII restriction enzymes. Biotyping assigned 14 strains as Lactobacillus casei, 6 strains as Lactobacillus paracasei subsp. paracasei and 12 as Lactobacillus rhamnosus. The obtained ribotype patterns separated all analyzed strains into two clearly distinguished groups corresponding to L. rhamnosus and L. casei/L. paracasei subsp. paracasei. The HindIII ribotypes of individual strains representing these two groups were visually very similar. In contrast, EcoRI ribotyping revealed high intraspecies variability. All ribotypes of L. casei and L. paracasei subsp. paracasei dairy strains were very close and some strains even shared identical ribotype profiles. The type strains L. casei CCM 7088T (= ATCC 393T) and Lactobacillus zeae CCM 7069T revealing similar ribopatterns formed a separate subcluster using both restriction enzymes. In contrast, the ribotype profile of L. casei CCM 7089 (= ATCC 334) was very close to ribopatterns obtained from the dairy strains. These results support synonymy of L. casei and L. paracasei species revealed by other studies as well as reclassification of the type strain L. casei CCM 7088T as L. zeae and designation of L. casei CCM 7089 as the neotype strain.     

Effects of Lactobacillus casei on Pseudomonas aeruginosa infection in normal and dexamethasone-treated mice

A single intraperitoneal injection of Lactobacillus casei YIT 0003 into normal or dexamethasone-treated mice led to nonspecific resistance against intraperitoneal challenge with lethal doses of Pseudomonas aeruginosa PAO 3047. The enhanced resistance was retained for 14 days (P less than 0.05) after injection with living L. casei. In contrast, the statistically significant duration of the enhanced resistance in mice treated intraperitoneally with living L. acidophilus YIT 0075 was only 5 days. The in vivo killing activity of peritoneal exudate cells (PECs) against P. aeruginosa 5 and 7 days after intraperitoneal injection of living L. casei was significantly higher than in the case of PECs elicited by L. acidophilus. In the case of intravenous injection of heat-killed L. casei before intraperitoneal challenge with P. aeruginosa, there were no survivors in the late period after administration of L. casei. A high correlation existed between the patterns of in vivo killing of P. aeruginosa by PECs and survival rate of mice injected intravenously with heat-killed L. casei. The reduced in vivo killing activity of PECs from dexamethasone-treated mice against P. aeruginosa infection was also augmented by the intraperitoneal injection of heat-killed L. casei. These results indicate that L. casei possesses a resistance-enhancing capacity against P. aeruginosa infection in vivo. Differences in the duration of the enhanced resistance caused by L. casei and by L. acidophilus may be due to differences in chemical composition and/or physicochemical properties of the cell walls of the two kinds of bacteria.     

Characterization of the DNA binding region recognized by dihydrofolate reductase from lactobacillus casei

Two specific DNA binding sites for the enzyme dihydrofolate reductase from Lactobacillus casei have been located by means of an immunoprecipitation assay within a 2900-base pair L. casei DNA fragment containing the L. casei dihydrofolate reductase structural gene, which was previously cloned into pBR322. The inserted L. casei DNA was mapped using restriction endonucleases, and the location and orientation of the structural gene coding for L. casei dihydrofolate reductase were determined. The two specific binding sites map at the 5' end of the structural gene, approximately 100 base pairs upstream from the start of the coding region.     

Shuttle plasmid vectors for Lactobacillus casei and Escherichia coli with a minus origin.

Recombinant plasmids which can be used as shuttle vectors between Escherichia coli and the industrially used strains of Lactobacillus casei were constructed. They have replication regions closely related to those of pUB110 and are likely to replicate by a rolling-circle mechanism via a plus-strand-specific DNA intermediate in L. casei. Both orientations of palA from the staphylococcal plasmid pC194 and those of the intergenic region from coliphage M13 are identified as active minus origins in L. casei, in contrast to the pAM alpha 1 delta 1-derived BA3 minus origin which does not function in L. casei. Stability of the plasmids increased in L. casei when one of these two active minus origins was inserted. All the DNA sequences of the constructed vectors were known.     

表面展示新城疫病毒部分F蛋白的重组干酪乳杆菌的构建及其免疫原性

本研究旨在构建重组干酪乳杆菌pLA-Newcastlediseasevirus (NDV)-F/Lactobacillus casei,获得表达产物,并探讨其免疫效果。利用PCR扩增携带部分主要抗原表位的NDV F基因,与穿梭质粒pLA连接转化至大肠杆菌BL21 (DE3)中,筛选阳性重组质粒,将其电转化至干酪乳杆菌中,构建重组干酪乳杆菌pLA-NDV-F/L. casei,应用PCR鉴定阳性菌株,Western blotting鉴定重组菌反应原性,间接免疫荧光、流式细胞术和激光共聚焦检测蛋白表达情况。试验选用14日龄雏鸡,各组免疫方式为口服+滴鼻。设立pLA-NDV-F/L. casei两次免疫组和三次免疫组、弱毒疫苗组、 pLA/L.casei、未攻毒PBS组和攻毒PBS组。间接ELISA方法检测雏鸡血清IgG、肠道、鼻腔、肺脏中sIgA抗体效价,评价试验组雏鸡攻毒保护率。结果表明,有94.10%的重组菌表达了F蛋白,且高效表达在干酪乳杆菌细胞表面,蛋白大小为62kDa,并能与抗NDV阳性血清特异性结合。各免疫组anti-F IgG和s Ig A抗体水平显著高于对照组,p LA-NDV-F/L. casei三次免疫组抗体持续时间比两次免疫组延长28 d,抗体峰值没有显著差异。免疫pLA-NDV-F/L. casei三次、两次、弱毒疫苗、pLA/L. casei和PBS的攻击保护率分别为80%、80%、90%、0%和0%。因此,利用干酪乳杆菌表达体系成功表达了携带部分抗原表位的NDVF基因,具备良好的反应原性和免疫原性,可诱导机体产生保护性免疫应答。     

Some peculiarities of functioning of H+-ATPase from the membranes of the anaerobic bacterium Lactobacillus casei

Radiation inactivation analysis gave the target sizes of 176 +/- 5 kDa and 275 +/- 33 kDa for ATPase from anaerobic Lactobacillus casei and aerobic Micrococcus luteus bacteria respectively. The values are close to the known molecular masses of the enzymes. Thus, to function the L. casei ATPase, like the F1-ATPases, requires a complete structure composed of all the enzyme subunits. L. casei ATPase is inhibited by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole owing to modification of an amino acid residue(s) with pK greater than 8.5. L. casei ATPase consists of six identical subunits and differs from alpha 3 beta 3 gamma delta epsilon-type F1-ATPases in a number of catalytic properties. Namely, ATP hydrolysis under the 'unisite' conditions proceeds at a relatively high rate suggesting the absence of cooperative interactions between the catalytic sites. Contrary to mitochondrial F1-ATPase. L. casei ATPase does not form an inactive complex with ADP. These findings imply essential differences in the operating mechanism for L. casei ATPase and F1 ATPase.     

Molecular cloning and nucleotide sequence of the factor IIIlac gene of Lactobacillus casei

The lactose-specific factor III (FIIIlac of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Lactobacillus casei and purified to homogeneity by conventional protein purification methods. Its apparent native Mr, estimated from steric exclusion chromatography (approx. 39 kDa), and subunit Mr, estimated from sodium dodecyl sulfate-polyacrylamide gels, indicated that it exists as a trimer of identical subunits of 13 kDa. The gene for FIII L. casei lac was cloned into Escherichia coli using the vector pUC18. The coding sequences were contained on an 860-bp BglII-HindIII DNA fragment of the L. casei lactose plasmid, pLZ64. A protein identical in properties to FIII L. casei lac was isolated from clones of E. coli carrying this DNA insert. The nucleotide sequence of the FIII L. casei lac gene was determined by the dideoxy chain-termination technique. The 336-bp open reading frame for FIII L. casei lac was followed by a stem-loop structure, analogous to a Rho-independent terminator. We concluded that the FIII L. casei lac was the terminal gene in what appears to be an operon comprised of the lactose-PTS-P-beta Gal-coding genes. Comparison of the deduced amino acid sequence of FIII L. caseilac with the amino acid sequence of FIII S. aureus lac (derived from peptide sequencing) demonstrated a high degree of homology (49 identical residues and 21 conservative exchanges out of 103 total aa residues). The FIII L. casei lac lacked his82, previously identified as the phosphorylation site of FIII S. aureus. lac His80 was proposed to be the site of histidyl phosphorylation of FIII L. casei lac.