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Independent insertion of Alu elements in the human ribosomal spacer and their concerted evolution.

A 2,700-bp segment of human ribosomal DNA (rDNA) spacer upstream of the rRNA promoter contains a set of four Alu elements, two in the direction of rRNA transcription and two in the opposite orientation. We report and compare the sequences of these Alu elements found in three rDNA clones and seek to determine the origin of the cluster, either from a single insertion followed by duplications or from multiple simultaneous or independent insertions. The high (20%-27%) divergence among members of a set and the lack of similarity/complementarity of sequences flanking different members of the set demonstrate the independent insertion of each of the four Alu elements into A-rich sequences on the appropriate strand of the rDNA. We also demonstrate that the Alu sets found in different rDNA repeats are subject to concerted evolution, yielding divergences of only 0.4%-3% between Alu elements in equivalent positions. However, the pairs of adjacent similarly oriented Alu elements do not show reduced divergence, indicating that there is no recombination or gene conversion between similarly oriented but not equivalently positioned Alu elements. Finally, crossing-over must occur in the rDNA junction region between Alu element 3 and the nonribosomal sequences at the telomere end of the acrocentric chromosome, so that the Alu elements of the terminal rDNA repeats and the terminal repeats themselves evolve in concert with the rDNA repeats located internally in the tandem array.

Base Sequence↗

Distribution of hobo transposable elements in natural populations of Drosophila melanogaster.

Forty-six strains derived from American and French natural populations of Drosophila melanogaster were tested for the presence and activity of hobo elements by using Southern blotting and a gonadal dysgenesis assay. The oldest available strains exhibited weak detectable hybridization to the hobo-element probe and revealed neither hobo-activity potential nor hobo-repression potential. In contrast, all recently collected strains harbored hobo sequences and revealed a strong hobo-repression potential but no strong hobo-activity potential. On the basis of restriction-enzyme analysis, old strains appear to have numerous fragments hybridizable to hobo sequences, several probably conserved at the same locations in the genome of the tested strain and others dispersed. In recently isolated strains, and unlike the situation in the published sequence of the cloned hobo108 element, a PvuII site is present in the great majority of full-sized hobo elements and their deletion derivatives. When the genetic and molecular characteristics are considered together, the available evidence is consistent with the hypothesis of a worldwide hobo-element invasion of D. melanogaster during the past 50 years. Comparison of data from the I-R and P-M systems suggests that the putative invasion followed the introduction of the I element but preceded that of the P element. This hypothesis poses the problem of the plausibility of three virtually simultaneous element invasions in this species. Such a possibility might be due to a modification of the genetic structure of American populations of D. melanogaster during the first part of the 20th century.

Americas↗

Trace element nutrition during pregnancy.

The best means to ensure an optimal intake of trace elements during pregnancy is the consumption of a well-balanced diet that includes both animal- and plant-food sources. Although vegetarian diets provide reasonable sources of trace elements, especially in dried beans and seeds, flesh foods contain higher concentrations of trace elements that are in a more readily absorbable form. Iron is the only trace element for which routine supplementation is recommended. In the United States, the iodine content of the food supply is sufficiently high to make supplementation unnecessary, but use of iodized salt is not contraindicated. There is no need to screen pregnant women routinely for trace element status, except to monitor hemoglobin and hematocrit as an indicator of iron status. The best indicators that a woman's trace element status may be at risk is a history of poor food selections, a clinical disorder that alters trace element use or excretion, or prior residence in a region of the world where the trace element content of the soil is low.

Female↗

Nucleotide sequence of a repetitive element isolated from Leptospira interrogans serovar hardjo type hardjo-bovis.

A repetitive element from the genome of Leptospira interrogans serovar hardjo type hardjo-bovis ('L. hardjo-bovis') was identified, cloned and sequenced. Similar sequences were shown by hybridization to be encoded by a further eight of 32 other leptospiral serovars tested. An undefined number of repetitive elements were located in the L. hardjo-bovis genome; sequence degeneracy of the elements was observed and no significant open reading frames were identified within the AT-rich (60%) 1467 bp repetitive element. The termini encoded a GC-rich 8 bp repeat motif and two variants showed rearrangements centred on these motifs. The nucleotide sequences of the chromosomal regions flanking the repetitive elements were determined but showed no similarities, with one exception which had a GAAC repeat directly adjacent to both termini. Similar hybridization patterns were shown by Southern transfers of L. hardjo-bovis total genomic digests probed with the repetitive element. Oligonucleotide primer pairs designed from sequences internal to the repetitive element and adjacent chromosomal regions were used in polymerase chain reaction experiments. With one primer pair all L. hardjo-bovis isolates, but no other serovar, gave identical amplified products. Evidence that the repetitive element may have derived from an acquired insertion sequence that is now inactive and chromosomally fixed is discussed.

Base Sequence↗

An endocrine-specific element is an integral component of an exocrine-specific pancreatic enhancer.

We have analyzed the function of individual elements of the elastase I transcriptional enhancer in transgenic animals. This pancreas-specific enhancer comprises three functional elements, one of which (the B element) plays a dual role. Within the context of the enhancer, the B element contributes to appropriate acinar cell expression. However, when separated from the other enhancer components, the B element selectively directs transcription in islet cells of transgenic animals. This islet-specific activity is normally suppressed by an upstream repressor domain. The B element binds a novel islet-specific factor, and similar B-like elements are present in other pancreatic genes, both exocrine and endocrine specific. We suggest that a principal role of this transcriptional element and its associated factors is to activate many pancreatic genes as part of the program of pancreatic determination prior to the divergence of the acinar and islet cell lineages.

Amylases↗

HnRNP L binds a cis-acting RNA sequence element that enables intron-dependent gene expression.

Most pre-mRNAs require an intron for efficient processing in higher eukaryotes. To test the hypothesis that intron-independent gene expression involves positive, cis-acting RNA sequence elements, we constructed chimeric genes in which various regions of the naturally intronless HSV-TK gene were inserted into an intronless variant of the highly intron-dependent human beta-globin gene. Using a transient transfection assay, we identified a 119-nucleotide sequence element contained within the transcribed region of the HSV-TK gene that enables efficient cytoplasmic accumulation of globin RNA in the absence of splicing. RNA UV-cross-linking assays indicated that a 68-kD protein present in nuclear extracts of HeLa and COS cells specifically binds to this HSV-TK sequence element. This 68-kD protein was found to cross-react with an antiserum specific to hnRNP L. Recombinant hnRNP L was shown to bind with high sequence specificity to this RNA sequence element. Analysis of substitution mutants in this element indicated that binding of hnRNP L correlates with accumulation of the RNA in the cytoplasm. Thus, we conclude that (1) hnRNP L binds in a sequence-specific manner to this RNA sequence element that enables intron-independent gene expression, and (2) intron-independent pre-mRNA processing and transport involves sequence-specific RNA-protein interactions between cis-acting RNA sequence elements and proteins such as hnRNP L. This sequence element may be of general use for the efficient expression of cDNA versions of intron-dependent genes.

Animals↗

Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods.

We report here the identification of a previously unknown transcription regulatory element for heat shock (HS) genes in Caenorhabditis elegans. We monitored the expression pattern of 11,917 genes from C. elegans to determine the genes that were up-regulated on HS. Twenty eight genes were observed to be consistently up-regulated in several different repetitions of the experiments. We analyzed the upstream regions of these genes using computational DNA pattern recognition methods. Two potential cis-regulatory motifs were identified in this way. One of these motifs (TTCTAGAA) was the DNA binding motif for the heat shock factor (HSF), whereas the other (GGGTGTC) was previously unreported in the literature. We determined the significance of these motifs for the HS genes using different statistical tests and parameters. Comparative sequence analysis of orthologous HS genes from C. elegans and Caenorhabditis briggsae indicated that the identified DNA regulatory motifs are conserved across related species. The role of the identified DNA sites in regulation of HS genes was tested by in vitro mutagenesis of a green fluorescent protein (GFP) reporter transgene driven by the C. elegans hsp-16-2 promoter. DNA sites corresponding to both motifs are shown to play a significant role in up-regulation of the hsp-16-2 gene on HS. This is one of the rare instances in which a novel regulatory element, identified using computational methods, is shown to be biologically active. The contributions of individual sites toward induction of transcription on HS are nonadditive, which indicates interaction and cross-talk between the sites, possibly through the transcription factors (TFs) binding to these sites.

Animals↗

A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome.

We transposed Dissociation (Ds) elements from three start loci on chromosome 5 in Arabidopsis (Nossen ecotype) by using a local transposition system. We determined partial genomic sequences flanking the Ds elements and mapped the elements' insertion sites in 1,173 transposed lines by comparison with the published genomic sequence. Most of the lines contained a single copy of the Ds element. One-half of the lines contained Ds on chromosome 5; in particular, insertion "hot spots" near the three start loci were clearly observed. In the other lines, the Ds elements were transposed across chromosomes. We found other insertion hot spots at the tops of chromosomes 2 and 4, near nucleolus organizer regions 2 and 4, respectively. Another characteristic feature was that the Ds elements tended to transpose near the chromosome ends and rarely transposed near centromeres. The distribution patterns differed among the three start loci, even though they possessed the same Ds construct. More than one-half of the Ds elements were inserted irregularly into the genome; that is, they did not retain the perfect inverted repeat sequence of Ds nor leave perfect target site duplications. This precise analysis of distribution patterns will contribute to a comprehensive understanding of the transposing mechanism. From these Ds insertion sites, we have constructed a database for screening gene-knockout mutants in silico. In 583 of the 1,173 lines, the Ds elements were inserted into protein-coding genes, which suggests that these lines are gene-knockout mutants. The database and individual lines will be available freely for academic use from the RIKEN Bio-Resource Center (http://www.brc.riken.go.jp/Eng/index.html).

Arabidopsis↗

Dasheng and RIRE2. A nonautonomous long terminal repeat element and its putative autonomous partner in the rice genome.

Dasheng is one of the highest copy number long terminal repeat elements and one of the most recent elements to amplify in the rice (Oryza sativa) genome. However, the absence of any significant coding capacity for retroviral proteins, including gag and pol, suggests that Dasheng is a nonautonomous element. Here, we have exploited the availability of 360 Mb of rice genomic sequence to identify a candidate autonomous element. RIRE2 is a previously described gypsy-like long terminal repeat retrotransposon with significant sequence similarity to Dasheng in the regions where putative cis factors for retrotransposition are thought to be located. Dasheng and RIRE2 elements have similar chromosomal distribution patterns and similar target site sequences, suggesting that they use the same transposition machinery. In addition, the presence of several RIRE2-Dasheng element chimeras in the genome is consistent with the copackaging of element mRNAs in the same virus-like particle. Finally, both families have recently amplified members, suggesting that they could have been co-expressed, a necessary prerequisite for RIRE2 to serve as the source of transposition machinery for Dasheng. Consistent with this hypothesis, transcripts from both elements were found in the same expressed sequence tag library.

Base Sequence↗

Analyses of P-like transposable element sequences from the genome of Anopheles gambiae.

We have identified 50 P element-homologous sequences in the genome of Anopheles gambiae by performing homology searches against the public genome database of A. gambiae using the canonical P element from Drosophila melanogaster as a query sequence. While most of these sequences belong to P subfamilies previously described from anopheline mosquitoes, at least four new subfamilies were identified. One of these A. gambiae P elements, which we termed AgPLS, was analysed in detail. AgPLS consists of three exons and does not have inverted terminal repeats. This element retains several of the structural features of other P-encoded peptides, such as motifs involved in DNA-protein and protein-protein interaction, and a motif involved in GTP utilization. Strong sequence and structural similarity to functional P elements, a number of nonsynonymous substitutions that is smaller than that of synonymous substitutions and the presence of putative nuclear localization signals suggest that the A. gambiae elements may retain the capacity for transposition or its repression. These sequences seem to be most closely related to P elements described from Musca domestica and Lucilia cuprina, the only P element hosts known outside the family Drosophilidae.

Amino Acid Sequence↗

Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila.

We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.

Blotting, Southern↗

Modulation of the mtrCDE-encoded efflux pump gene complex of Neisseria meningitidis due to a Correia element insertion sequence.

The mtr (multiple transferable resistance) gene complex in Neisseria gonorrhoeae encodes an energy-dependent efflux pump system that is responsible for export of anti-bacterial hydrophobic agents. Expression of the mtrCDE operon in gonococci is negatively regulated by the MtrR protein. Hydrophobic agent resistance mediated by the mtr system is also inducible, which results from an AraC-like protein termed MtrA. In this work, we identified and characterized a pump similar to the gonococcal mtr system in various strains of Neisseria meningitidis. Unlike the situation with gonococci, the mtr system in meningococci is not subject to the MtrR or MtrA regulatory schemes. An analysis of the promoter region of the mtrCDE operon in a panel of meningococcal strains revealed the presence of one or two classes of insertion sequence elements. A 155-159 bp insertion sequence element known as the Correia element, previously identified elsewhere in the gonococcal and meningococcal genomes, was present in the mtrCDE promoter region of all meningococcal strains tested. In addition to the Correia element, a minority of strains had a tandemly linked, intact copy of IS1301. As described previously, a binding site for the integration host factor (IHF) was present at the centre of the Correia element upstream of mtrCDE genes. IHF was found to bind specifically to this site and deletion of the IHF binding site enhanced mtrC transcription. We also identified a post-transcriptional regulation of the mtrCDE transcript by cleavage in the inverted repeat of the Correia element, as previously described by Mazzone et al. [Gene278: 211-222 (2001)] and De Gregorio et al. [Biochim Biophys Acta 1576: 39-44 (2002)]for other Correia element. We conclude that the mtr efflux system in meningococci is subject to transcriptional regulation by IHF and post-transcriptional regulation by cleavage in the inverted repeat of the Correia element.

Amino Acid Sequence↗

Effect of toothbrushing on elemental release from dental casting alloys.

PURPOSE: This study assessed the effect of toothbrushing on the elemental release from common dental casting alloys. MATERIALS AND METHODS: A Au-Pt high-noble alloy, a Au-Pd high-noble alloy, a Pd-Cu-Ga noble alloy, and a Ni-Cr base-metal alloy were placed into a biological medium for 1 week to allow equilibration of elemental release, then brushed for 30 minutes at 90 strokes/min under 200 g of force with a soft toothbrush. The brushing was performed in neutral saline or lactic acid at pH 4 with or without toothpaste. The alloys were then transferred back into a biological medium for 1 week. Atomic absorption spectroscopy was used to measure the mass lost from each alloy during and in the week after brushing. Mass loss was expressed as micrograms per square centimeter of alloy surface, and the various brushing conditions were compared by analysis of variance and Tukey multiple comparison intervals (alpha = 0.05). RESULTS: During brushing, element release increased significantly over nonbrushed controls for all alloys except the Ni-Cr. Toothpastes, acidic solutions, or combinations of these conditions with toothbrushing further enhanced elemental release, but Ni release from the Ni-Cr alloy increased the most (30-fold). In the week after brushing, brushing alone increased elemental release only from the Ni-Cr alloy. However, toothpastes and acidic solutions increased elemental release for all alloys significantly, except for the Au-Pd alloy. In general, elemental release during brushing was far greater than in the week after brushing. CONCLUSIONS: Under the conditions of this study, toothbrushing increased the elemental release from dental casting alloys.

Analysis of Variance↗

A novel ICE in the genome of Shewanella putrefaciens W3-18-1: comparison with the SXT/R391 ICE-like elements.

A novel R391-like ICE (integrating conjugative element) has been detected in the 4.2 MB genome of Shewanella putrefaciens W3-18-1 located on three different contigs. Assembly of the ICE encoding contigs based on similarity with R391 revealed a mosaic element of plasmid, phage and transposon-like sequences typical of SXT/R391 ICE-like elements. The element, which is 110 057 bp in length, was highly similar to R391 sequences, with most related ORFs showing >96% amino acid sequence identity. The element, designated ICESpuPO1, contained a number of inserts determining resistance to copper and other heavy metals and a broad-spectrum RND efflux pump similar to antibiotic efflux systems. The element was integrated into the Shewanella prfC gene in a manner similar to related ICE-like elements. The chromosomal element junctions contained a 17-bp SXT/R391-like attL and attR site and an unannotated ORF between attL and the ICE integrase encoding a putative recombinational directional factor necessary for excision, with 100% amino acid identity to the R391 ORF4 product.

Bacterial Proteins↗

Roles of salicylic acid-responsive cis-acting elements and W-boxes in salicylic acid induction of VCH3 promoter in transgenic tobaccos.

A salicylic acid (SA)-inducible VCH3 promoter was recently identified from grapevine (Vitis amurensis) that contains two inverse SA-responsive cis-acting elements and four W-boxes. To further demonstrate the roles of these elements, four fragments with lengths from -1187, -892, -589, -276 to +7 bp were fused with the b-glucuronidase (GUS) reporter gene and transferred to Nicotiana tobacum, together with another four VCH3 promoter fragments with mutation in the two inverse SA-responsive elements. The functions of each promoter fragment were examined by analysis of GUS activity in the transgenic tobacco root treated with SA. Enhanced GUS activity was shown in the roots of transgenic tobaccos with the VCH3 (-1187)-GUS construct containing two SA-responsive cis-acting elements and four W-boxes. However, GUS activity directed by the VCH3 (-892)-GUS construct, containing one SA cis-acting element and four W-boxes, was reduced by up to 35% compared with that in tobaccos transformed with the VCH3 (-1187)-GUS construct, indicating that the SA cis-acting element plays an important role in SA induction of the VCH3 promoter. Neither the m2VCH3 (-1187)-GUS nor the mVCH3 (-892)-GUS construct, with mutation on the SA-responsive elements, abolished the expression of GUS activity, demonstrating that the W-boxes in the VCH3 promoter are also involved in SA induction. Histochemical analysis of GUS activity directed by each of the eight VCH3 promoter fragments showed that GUS was expressed specifically in vascular tissue. It was concluded that both the SA-responsive cis-acting elements and the W-boxes are important for the SA induction of the VCH3 promoter. This promoter might have a potential use in plant genetic engineering.

Gene Deletion↗

Multiple genetic elements carry the tetracycline resistance gene tet(W) in the animal pathogen Arcanobacterium pyogenes.

The tet(W) gene is associated with tetracycline resistance in a wide range of bacterial species, including obligately anaerobic rumen bacteria and isolates from the human gut and oral mucosa. However, little is known about how this gene is disseminated and the types of genetic elements it is carried on. We examined tetracycline-resistant isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes, all of which carried tet(W), and identified three genetic elements designated ATE-1, ATE-2, and ATE-3. These elements were found in 25%, 35%, and 60% of tetracycline-resistant isolates, respectively, with some strains carrying both ATE-2 and ATE-3. ATE-1 shows characteristics of a mobilizable transposon, and the tet(W) genes from strains carrying this element can be transferred at low frequencies between A. pyogenes strains. ATE-2 has characteristics of a simple transposon, carrying only the resistance gene and a transposase, while in ATE-3, the tet(W) gene is associated with a streptomycin resistance gene that is 100% identical at the DNA level with the aadE gene from the Campylobacter jejuni plasmid pCG8245. Both ATE-2 and ATE-3 show evidence of being carried on larger genetic elements, but conjugation to other strains was not observed under the conditions tested. ATE-1 was preferentially associated with A. pyogenes strains of bovine origin, while ATE-2 and ATE-3 elements were primarily found in porcine isolates, suggesting that these elements may circulate in different environments. In addition, four alleles of the tet(W) gene, primarily associated with different elements, were detected among A. pyogenes isolates.

Actinomycetaceae↗

New insertion sequence elements in the upstream region of cfiA in imipenem-resistant Bacteroides fragilis strains.

The 747-bp cfiA gene, which encodes a metallo-beta-lactamase, and the regions flanking cfiA in six imipenem-resistant and four imipenem-susceptible Bacteroides fragilis strains isolated in Japan were analyzed by PCR and DNA sequencing. The nucleotide sequences of the cfiA genes (designated cfiA(1) to cfiA(10)) of all 10 strains tested varied from that of the standard cfiA gene from B. fragilis TAL2480. However, putative proteins encoded by the cfiA variants contained conserved amino acid residues important for zinc binding and hairpin loop formation, suggesting that cfiA variants have the capability of producing metallo-beta-lactamases with full catalytic activities. PCR assay indicated that six metallo-beta-lactamase-producing, imipenem-resistant strains had an insertion mutation in the region immediately upstream of cfiA. Nucleotide sequencing of the PCR-amplified fragments along with the upstream region of cfiA revealed that there were five new kinds of insertion sequence (IS) elements (designated IS612, IS613, IS614, IS615, and IS616, with a size range of 1,594 to 1,691 bp), of which only IS616 was found to be almost identical to IS1188, one of the IS elements previously identified in the upstream region of cfiA. These elements had target site duplications of 4 or 5 bp in length, terminal inverted repeats (14, 15, or 17 bp in size), and a large open reading frame encoding a putative transposase which is required for the transcription of IS elements. Each element was inserted such that the transcriptional direction of the transposase was opposite to that of cfiA. A computer-aided homology search revealed that, based on the homology of their putative transposases, the sizes of their terminal inverted repeat sequences, and their target site duplications, IS612, IS613, IS614, and IS615 belong to the IS4 family, which includes IS942, previously found in some drug-resistant B. fragilis strains, but that IS616 belongs to the IS1380 family. All the IS elements appear to have putative promoter motif sequences (the -7 region's TAnnTTTG motif and the -33 region's TTG or TG) in their end regions, suggesting that the IS elements provide a promoter for the transcription of cfiA upon insertion. These data provide additional proof that various IS elements may exist to provide a promoter to express the cfiA gene.

Amino Acid Sequence↗

ISLpl1 is a functional IS30-related insertion element in Lactobacillus plantarum that is also found in other lactic acid bacteria.

We describe the first functional insertion sequence (IS) element in Lactobacillus plantarum. ISLpl1, an IS30-related element, was found on the pLp3 plasmid in strain FB335. By selection of spontaneous mutants able to grow in the presence of uracil, it was demonstrated that the IS had transposed into the uracil phosphoribosyltransferase-encoding gene upp on the FB335 chromosome. The plasmid-carried IS element was also sequenced, and a second potential IS element was found: ISLpl2, an IS150-related element adjacent to ISLpl1. When Southern hybridization was used, the copy number and genome (plasmid versus chromosome) distribution data revealed different numbers and patterns of ISLpl1-related sequences in different L. plantarum strains as well as in Pediococcus strains. The ISLpl1 pattern changed over many generations of the strain L. plantarum NCIMB 1406. This finding strongly supports our hypothesis that ISLpl1 is a mobile element in L. plantarum. Database analysis revealed five quasi-identical ISLpl1 elements in Lactobacillus, Pediococcus, and Oenococcus strains. Three of these elements may be cryptic IS, since point mutations or 1-nucleotide deletions were found in their transposase-encoding genes. In some cases, ISLpl1 was linked to genes involved in cold shock adaptation, bacteriocin production, sugar utilization, or antibiotic resistance. ISLpl1 is transferred among lactic acid bacteria (LAB) and may play a role in LAB genome plasticity and adaptation to their environment.

Bacterial Proteins↗