16S Ribosomal DNA Terminal Restriction Fragment Pattern Analysis of Bacterial Communities in Feces of Rats Fed Lactobacillus acidophilus NCFM

16S ribosomal DNA terminal restriction fragment patterns from rat fecal samples were analyzed to track the dynamics of Lactobacillus acidophilus NCFM and discern bacterial populations that changed during feeding with NCFM. Lactobacillus johnsonii and Ruminococcus flavefaciens were tentatively identified as such bacterial populations. The presence of L. johnsonii was confirmed by isolation from feces.     

Group-specific comparison of four lactobacilli isolated from human sources using differential blast analysis

Lactic acid bacteria (LAB) have been used in fermentation processes for centuries. More recent applications including the use of LAB as probiotics have significantly increased industrial interest. Here we present a comparative genomic analysis of four completely sequenced Lactobacillus strains, isolated from the human gastrointestinal tract, versus 25 lactic acid bacterial genomes present in the public database at the time of analysis. Lactobacillus acidophilus NCFM, Lactobacillus johnsonii NCC533, Lactobacillus gasseri ATCC33323, and Lactobacillus plantarum WCFS1are all considered probiotic and widely used in industrial applications. Using Differential Blast Analysis (DBA), each genome was compared to the respective remaining three other Lactobacillus and 25 other LAB genomes. DBA highlighted strain-specific genes that were not represented in any other LAB used in this analysis and also identified group-specific genes shared within lactobacilli. Initial comparative analyses highlighted a significant number of genes involved in cell adhesion, stress responses, DNA repair and modification, and metabolic capabilities. Furthermore, the range of the recently identified potential autonomous units (PAUs) was broadened significantly, indicating the possibility of distinct families within this genetic element. Based on in silico results obtained for the model organism L. acidophilus NCFM, DBA proved to be a valuable tool to identify new key genetic regions for functional genomics and also suggested re-classification of previously annotated genes.     

Indication for Co-evolution of Lactobacillus johnsonii with its hosts

ABSTRACT: BACKGROUND: The intestinal microbiota, composed of complex bacterial populations, is host-specific and affected by environmental factors as well as host genetics. One important bacterial group is the lactic acid bacteria (LAB), which include many health-promoting strains. Here, we studied the genetic variation within a potentially probiotic LAB species, Lactobacillus johnsonii, isolated from various hosts. RESULTS: A wide survey of 104 fecal samples was carried out for the isolation of L. johnsonii. As part of the isolation procedure, terminal restriction fragment length polymorphism (tRFLP) was performed to identify L. johnsonii within a selected narrow spectrum of fecal LAB. The tRFLP results showed host specificity of two bacterial species, the Enterococcus faecium species cluster and Lactobacillus intestinalis, to different host taxonomic groups while the appearance of L. johnsonii and E. faecalis was not correlated with any taxonomic group. The survey ultimately resulted in the isolation of L. johnsonii from few host species The genetic variation among the 47 L. johnsonii strains isolated from the various hosts was analyzed based on variation at simple sequence repeats (SSR) loci and multi-locus sequence typing (MLST) of conserved hypothetical genes. The genetic relationships among the strains inferred by each of the methods were similar, revealing three different clusters of L. johnsonii strains, each cluster consisting of strains from a different host, i.e., chickens, humans or mice. CONCLUSIONS: Our typing results support phylogenetic separation of L. johnsonii strains isolated from different animal hosts, suggesting specificity of L. johnsonii strains to their hosts. Taken together with the tRFLP results, that indicated the association of specific LAB species with the host taxonomy, our study supports co-evolution of the host and its intestinal lactic acid bacteria.     

Predominant genera of fecal microbiota in children with atopic dermatitis are not altered by intake of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bi-07

The effect of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 on the composition of the Lactobacillus group, Bifidobacterium and the total bacterial population in feces from young children with atopic dermatitis was investigated. The study included 50 children randomized to intake of one of the probiotic strain or placebo. Microbial composition was characterized by denaturing gradient gel electrophoresis, quantitative PCR and, in a subset of subjects, by pyrosequencing of the 16S rRNA gene. The core population of the Lactobacillus group was identified as Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus oris, Leuconostoc mesenteroides, while the bifidobacterial community included Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum and Bifidobacterium catenulatum. The fecal numbers of L. acidophilus and B. lactis increased significantly after intervention, indicating survival of the ingested bacteria. The levels of Bifidobacterium correlated positively (P=0.03), while the levels of the Lactobacillus group negatively (P=0.01) with improvement of atopic eczema evaluated by the Severity Scoring of Atopic Dermatitis index. This correlation was observed across the whole study cohort and not attributed to the probiotic intake. The main conclusion of the study is that administration of L. acidophilus NCFM and B. lactis Bi-07 does not affect the composition and diversity of the main bacterial populations in feces.     

乳杆菌吸附塑化剂的影响因素分析及效果评价

【目的】评价嗜酸乳杆菌(Lactobacillus acidophilus) NCFM和类食品乳杆菌(Lactobacillus paralimentarius) 412吸附塑化剂的效果,初步探讨影响其吸附的因素。【方法】基于3种邻苯二甲酸酯类塑化剂：邻苯二甲酸二乙酯(DEP)、邻苯二甲酸二丁酯(DBP)和邻苯二甲酸二乙基己酯(DEHP)的HPLC检测方法,评价了菌株NCFM和412与塑化剂共同温育时的吸附状况,优化菌株吸附DBP的条件,研究菌株在热、酸及NaCl处理后吸附塑化剂的稳定性。【结果】乳杆菌NCFM和412对3种邻苯二甲酸酯类塑化剂均有不同程度的吸附效果,其中菌株NCFM对3种塑化剂DEP、DBP和DEHP的吸附率分别为21.48%、43.32%和9.62%,吸附效果优于菌株412,其中DBP的吸附效果最好。在温度为37 °C时,菌株NCFM吸附DBP的最佳时间是4 h。热、酸和NaCl处理都会显著提高菌体NCFM吸附塑化剂的效果。【结论】嗜酸乳杆菌NCFM通过吸附作用具有清除3种邻苯二甲酸酯类塑化剂的效果,可以作为潜在的塑化剂生物脱除剂使用。     

Similarity and differences in the Lactobacillus acidophilus group identified by polyphasic analysis and comparative genomics

A set of lactobacilli were investigated by polyphasic analysis. Multilocus sequence analysis, DNA typing, microarray analysis, and in silico whole-genome alignments provided a remarkably consistent pattern of similarity within the Lactobacillus acidophilus complex. On microarray analysis, 17 and 5% of the genes from Lactobacillus johnsonii strain NCC533 represented variable and strain-specific genes, respectively, when tested against four independent isolates of L. johnsonii. When projected on the NCC533 genome map, about 10 large clusters of variable genes were identified, and they were enriched around the terminus of replication. A quarter of the variable genes and two-thirds of the strain-specific genes were associated with mobile DNA. Signatures for horizontal gene transfer and modular evolution were found in prophages and in DNA from the exopolysaccharide biosynthesis cluster. On microarray hybridizations, Lactobacillus gasseri strains showed a shift to significantly lower fluorescence intensities than the L. johnsonii test strains, and only genes encoding very conserved cellular functions from L. acidophilus hybridized to the L. johnsonii array. In-silico comparative genomics showed extensive protein sequence similarity and genome synteny of L. johnsonii with L. gasseri, L. acidophilus, and Lactobacillus delbrueckii; moderate synteny with Lactobacillus casei; and scattered X-type sharing of protein sequence identity with the other sequenced lactobacilli. The observation of a stepwise decrease in similarity between the members of the L. acidophilus group suggests a strong element of vertical evolution in a natural phylogenetic group. Modern whole-genome-based techniques are thus a useful adjunct to the clarification of taxonomical relationships in problematic bacterial groups.     

A Chinese rhesus macaque (Macaca mulatta) model for vaginal Lactobacillus colonization and live microbicide development

Background  We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization suitable for evaluating live microbial microbicide candidates.
Methods  Vaginal and rectal microflora in Chinese rhesus macaques ( Macaca mulatta ) were analyzed, with cultivable bacteria identified by 16S rRNA gene sequencing. Live lactobacilli were intravaginally administered to evaluate bacterial colonization.
Results  Chinese rhesus macaques harbored abundant vaginal Lactobacillus , with Lactobacillus johnsonii as the predominant species. Like humans, most examined macaques harbored only one vaginal Lactobacillus species. Vaginal and rectal Lactobacillus isolates from the same animal exhibited different genetic and biochemical profiles. Vaginal Lactobacillus was cleared by a vaginal suppository of azithromycin, and endogenous L. johnsonii was subsequently restored by intravaginal inoculation. Importantly, prolonged colonization of a human vaginal Lactobacillus jensenii was established in these animals.
Conclusions  The Chinese rhesus macaque harbors vaginal Lactobacillus and is a potentially useful model to support the pre-clinical evaluation of Lactobacillus -based topical microbicides.     

嗜酸乳杆菌NCFM对Caco-2细胞中PTX3基因表达的影响

【目的】研究嗜酸乳杆菌NCFM对肠道上皮细胞中免疫与炎症介质因子PTX3表达的影响,并进一步揭示其调节机制。【方法】嗜酸乳杆菌NCFM与Caco-2细胞共培养0、2、4、8和12 h,提取细胞RNA,采用RealTime RT-PCR方法检测PTX3基因的表达。嗜酸乳杆菌NCFM与Caco-2细胞共培养0、0.5、1、2和4 h,提取细胞蛋白质,采用Western blot方法检测NF-κB的磷酸化水平;用NF-κB的特异性抑制剂PDTC预处理Caco-2细胞30 min,然后加入嗜酸乳杆菌NCFM作用2 h,提取细胞RNA,采用Real Time RT-PCR方法检测PTX3基因的表达。【结果】嗜酸乳杆菌NCFM与Caco-2细胞共培养后能诱导PTX3的表达,并且在共培养4 h的时候PTX3的表达量达到最大,然后逐渐下降;嗜酸乳杆菌NCFM能快速的诱导NF-κB的磷酸化,并且在加入其特异性抑制剂PDTC后,PTX3的表达显著下降。【结论】嗜酸乳杆菌NCFM作用于肠道上皮细胞后能够通过迅速激活NF-κB途径暂时性的调控PTX3的表达。     

Factors involved in adherence of lactobacilli to human Caco-2 cells.

A quantitative assay performed with bacterial cells labelled with [3H]thymidine was used to investigate factors involved in the adherence of human isolates Lactobacillus acidophilus BG2FO4 and NCFM/N2 and Lactobacillus gasseri ADH to human Caco-2 intestinal cells. For all three strains, adherence was concentration dependent, greater at acidic pH values, and significantly greater than adherence of a control dairy isolate, Lactobacillus delbrueckii subsp. bulgaricus 1489. Adherence of L. acidophilus BG2FO4 and NCFM/N2 was decreased by protease treatment of the bacterial cells, whereas adherence of L. gasseri ADH either was not affected or was enhanced by protease treatment. Putative surface layer proteins were identified on L. acidophilus BG2FO4 and NCFM/N2 cells but were not involved in adherence. Periodate oxidation of bacterial cell surface carbohydrates significantly reduced adherence of L. gasseri ADH, moderately reduced adherence of L. acidophilus BG2FO4, and had no effect on adherence of L. acidophilus NCFM/N2. These results indicate that Lactobacillus species adhere to human intestinal cells via mechanisms which involve different combinations of carbohydrate and protein factors on the bacterial cell surface. The involvement of a secreted bridging protein, which has been proposed as the primary mediator of adherence of L. acidophilus BG2FO4 in spent culture supernatant (M.-H. Coconnier, T. R. Klaenhammer, S. Kernéis, M.-F. Bernet, and A. L. Servin, Appl. Environ. Microbiol. 58:2034-2039, 1992), was not confirmed in this study. Rather, a pH effect on Caco-2 cells contributed significantly to the adherence of this strain in spent culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific identification and molecular typing analysis of Lactobacillus johnsonii by using PCR-based methods and pulsed-field gel electrophoresis

A fast and reliable Multiplex-PCR assay was established to identify the species Lactobacillus johnsonii. Two opposing rRNA gene-targeted primers have been designed for this specific PCR detection. Specificity was verified with DNA samples isolated from different lactic acid bacteria. Out of 47 Lactobacillus strains isolated from different environments, 16 were identified as L. johnsonii by PCR. The same set of strains was investigated with five alternative molecular typing methods: enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR), amplified fragment length polymorphism, single triplicate arbitrarily primed PCR, and pulsed-field gel electrophoresis in order to compare the discriminatory power of these methods. The reported data strongly support the highly significant heterogeneity among all L. johnsonii isolates, potentially linked to their origin of isolation. The use of species-specific primers as well as rapid and highly powerful PCR-based molecular typing tools (namely ERIC- and REP-PCR techniques) should be respectively envisaged for identifying, differentiating and monitoring L. johnsonii strains from various environmental samples, for product monitoring, for species tracing in clinical studies as well as bacterial profiling of various microecological or gastrointestinal environments.     

ESR spin trapping for characterization of radical formation in Lactobacillus acidophilus NCFM and Listeria innocua

In this study, radicals in pure cultures of Lactobacillus acidophilus NCFM and Listeria innocua were detected in a quantitative way by electron spin resonance spectroscopy using spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) or N-tert-butyl-α-phenylnitrone (PBN). No adverse effect of spin trap addition on viability was observed for any of the bacterial strains. L. acidophilus NCFM had a higher production of radicals than L. innocua when incubated in a growth medium. Furthermore, by using DMPO in a buffer system, the radicals produced by L. acidophilus NCFM could be identified as hydroxyl radicals. The presence of polyethylene glycol, impermeable for bacterial cells, decreased the signal intensity of the ESR spectrum of the DMPO–OH adduct in cultures of L. acidophilus NCFM and indicated quenching of hydroxyl radicals outside the bacteria. This suggests that radical production is an extracellular event for L. acidophilus NCFM.     

Species-specific oligonucleotide probes for the detection and identification of Lactobacillus isolated from mouse faeces

AIMS: To develop species-specific monitoring techniques for rapid detection and identification of Lactobacillus isolated from mouse faeces. METHODS AND RESULTS: The specificity of oligonucleotide probes was evaluated by dot blot hybridization to 16S rDNA and 23S rDNA amplified by PCR from 12 Lactobacillus type strains and 100 strains of Lactobacillus isolated from mouse faeces. Oligonucleotide probes specific for each Lactobacillus species hybridized only with targeted rDNA. The Lactobacillus strains isolated from mouse faeces were identified mainly as Lactobacillus intestinalis, L. johnsonii, L. murinus and L. reuteri using species-specific probes. 16S rDNA of eight unidentified isolates were sequenced and two new probes were designed. Four of eight strains of unhybridized Lactobacillus were identified as L. johnsonii/gasseri group, and the remaining four strains as L. vaginalis. CONCLUSIONS: The species-specific probe set of L. intestinalis, L. johnsonii, L. murinus, L. reuteri and L. vaginalis in this study was efficient for rapid identification of Lactobacillus isolated from mouse faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The oligonucleotide probe set for Lactobacillus species harboured in the mouse intestine, can be used for rapid identification of lactobacilli and monitoring of the faecal Lactobacillus community.     

嗜酸乳杆菌NCFM对Caco-2细胞基因表达谱的影响

摘要：【目的】嗜酸乳杆菌NCFM作为一株具有良好益生功能的模式菌株,采用基因芯片技术对其作用后的宿主细胞基因的变化情况进行分析,在生物、食品等领域具有较大研究价值。【方法】将嗜酸乳杆菌NCFM和Caco-2细胞共同培养2h,提取Caco-2细胞的总RNA,并将RNA反转录成cDNA,与Human Genome U133 Plus 2.0 Array基因表达谱芯片杂交,杂交后进行图像扫描和数据分析,并采用Real-time RT PCR方法对差异表达的基因进行验证。【结果】采用基因芯片方法检测了Caco-2细胞经嗜酸乳杆菌NCFM作用2h后的基因表达变化,发现差异表达基因为508个,其中有473个基因上调,35个基因下调,初步推测Caco-2细胞能诱导多个基因的表达,以发挥嗜酸乳杆菌NCFM的益生功能。并且经Real-time RT PCR验证,表达差异显著的3个免疫调节相关基因CCL2、PTX3和TNFRSF9的确在嗜酸乳杆菌NCFM作用期间高表达。【结论】以上这些结果促进了对嗜酸乳杆菌NCFM益生功能的认识,也为揭示该乳酸菌的作用机理提供了理论基础。     

Metabolic footprint of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> NCFM at different pH

Lactobacillus acidophilus NCFM is a well known microorganism from the genomic and probiotic point of view. In order to analyze the potential interactions of NCFM with the surrounding environment, in vitro tests with the metabolic footprinting approach were performed. It was found that NCFM increased the concentration of lactic acid, succinic acid, adenine and arginine in the medium. The metabolism of NCFM did not change significantly between pH 5 and 7, suggesting that other environmental factors than pH might have bigger impact on its colonization throughout the gastrointestinal tract.     

Analysis of the genome sequence of Lactobacillus gasseri ATCC 33323 reveals the molecular basis of an autochthonous intestinal organism

This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucus-binding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.     

Lactobacillus johnsonii La1 shares carbohydrate-binding specificities with several enteropathogenic bacteria

The carbohydrate-binding specificities of the probiotic lactic acid bacterium Lactobacillus johnsonii La1 (a health-beneficial bacterial strain able to be incorporated into the human intestinal microflora) were investigated in vitro. First various soluble complex carbohydrates were tested as potential inhibitors of the strain adhesion onto Caco-2 intestinal epithelial cells, and then bacterial binding to glycolipids immobilized on TLC plates was probed. Two major carbohydrate-binding specificities of Lactobacillus johnsonii La1 were identified. A first one for an Endo-H treated yeast cell wall mannoprotein carrying mainly O:-linked oligomannosides, and a second one for the gangliotri- and gangliotetra-osylceramides (asialo-GM1). Similar carbohydrate-binding specificities are known to be expressed on cell surface adhesins of several enteropathogens, enabling them to adhere to the host gut mucosa. These findings corroborate the hypothesis that selected probiotic bacterial strains could be able to compete with enteropathogens for the same carbohydrate receptors in the gut.     

Two-dimensional gel-based alkaline proteome of the probiotic bacterium Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM (NCFM) is a well-documented probiotic bacterium isolated from human gut. Detailed 2D gel-based NCFM proteomics addressed the so-called alkaline range, i.e., pH 6-11. Proteins were identified in 150 of the 202 spots picked from the Coomassie Brilliant Blue stained 2D gel using MALDI-TOF-MS. The 102 unique gene products among the 150 protein identifications were assigned to different functional categories, and evaluated by considering a calculated distribution of abundance as well as grand average of hydrophobicity values. None of the very few available lactic acid bacteria proteome reference maps included the range of pI >7.0. The present report of such data on the proteome of NCFM fundamentally complements current knowledge on protein profiles limited to the acid and neutral pH range.     

Predominant effect of host genetics on levels of Lactobacillus johnsonii bacteria in the mouse gut

The gut microbiota is strongly associated with the well-being of the host. Its composition is affected by environmental factors, such as food and maternal inoculation, while the relative impact of the host's genetics have been recently uncovered. Here, we studied the effect of the host genetic background on the composition of intestinal bacteria in a murine model, focusing on lactic acid bacteria (LAB) as an important group that includes many probiotic strains. Based on 16S rRNA gene genotyping, variation was observed in fecal LAB populations of BALB/c and C57BL/6J mouse lines. Lactobacillus johnsonii, a potentially probiotic bacterium, appeared at significantly higher levels in C57BL/6J versus BALB/c mouse feces. In the BALB/c gut, the L. johnsonii level decreased rapidly after oral administration, suggesting that some selective force does not allow its persistence at higher levels. The genetic inheritance of L. johnsonii levels was further tested in reciprocal crosses between the two mouse lines. The resultant F1 offspring presented similar L. johnsonii levels, confirming that mouse genetics plays a major role in determining these levels compared to the smaller maternal effect. Our findings suggest that mouse genetics has a major effect on the composition of the LAB population in general and on the persistence of L. johnsonii in the gut in particular. Concentrating on a narrow spectrum of culturable LAB enables the isolation and characterization of such potentially probiotic bacterial strains, which might be specifically oriented to the genetic background of the host as part of a personalized-medicine approach.     

In vivo characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry

AIMS: To test the efficacy of Lactobacillus johnsonii FI9785 in reducing the colonization and shedding of Salmonella enterica serotype Enteritidis, Escherichia coli O78:K80 and Clostridium perfringens in poultry. METHODS AND RESULTS: Specific pathogen-free chicks (1 day old) were dosed with a single oral inoculum of 1x10(9) CFU. Lactobacillus johnsonii FI9785 and 24 h later were challenged in separate experiments with S. Enteritidis (S1400, nalr) and E. coli O78:K80 (EC34195, nalr). There were no significant effects against S. Enteritidis whereas colonization of the small intestine by E. coli O78:K80 was reduced significantly. Both S. Enteritidis and E. coli colonized the caeca and colon to levels equivalent to control birds and there was no reduction in shedding as assessed by a semi-quantitative cloacal swabbing technique. Specific pathogen-free chicks (20 day old) were dosed with a single oral inoculum of 1x10(9) CFU L. johnsonii FI9785 and 24 h later were challenged with C. perfringens. A single oral dose of L. johnsonii FI9785 was sufficient to suppress all aspects of colonization and persistence of C. perfringens. CONCLUSIONS: Lactobacillus johnsonii FI9785 may be given to poultry for use as a competitive exclusion agent to control C. perfringens. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus johnsonii FI9785 may be a valuable tool to control the endemic disease of necrotic enteritis, thereby reducing economic losses associated with reduced use of antimicrobials in the poultry industry.     

Identification, characterisation and specificity of a cell wall lytic enzyme from Lactobacillus fermentum BR11

Screening of a genomic library with an antiserum raised against whole Lactobacillus fermentum BR11 cells identified a clone expressing an immunoreactive 37-kDa protein. Analysis of the 3010-bp DNA insert contained within the clone revealed four open reading frames (ORFs). One ORF encodes LysA, a 303 amino acid protein which has up to 35% identity with putative endolysins from prophages Lj928 and Lj965 from Lactobacillus johnsonii and Lp1 and Lp2 from Lactobacillus plantarum as well as with the endolysin of Lactobacillus gasseri bacteriophage Phiadh. The immunoreactive protein was shown to be encoded by a truncated ORF downstream of lysA which has similarity to glutamyl-tRNA synthetases. The N-terminus of LysA has sequence similarity with N-acetylmuramidase catalytic domains while the C-terminus has sequence similarity with putative cell envelope binding bacterial SH3b domains. C-terminal bacterial SH3b domains were identified in the majority of Lactobacillus bacteriophage endolysins. LysA was expressed in Escherichia coli and unusually was found to have a broad bacteriolytic activity range with activity against a number of different Lactobacillus species and against Lactococcus lactis, streptococci and Staphylococcus aureus. It was found that LysA is 2 and 8000 times more active against L. fermentum than L. lactis and Streptococcus pyogenes, respectively.