Chromosomal integration of Tn5253 occurs downstream of a conserved 11-bp sequence of the rbgA gene in Streptococcus pneumoniae and in all the other known hosts of this integrative conjugative element (ICE)

Abstract Background Tn5253, a composite Integrative Conjugative Element (ICE) of Streptococcus pneumoniae carrying tet(M) and cat resistance determinants, was found to (i) integrate at specific 83-bp integration site (attB), (ii) produce circular forms joined by a 84-bp sequence (attTn), and (iii) restore the chromosomal integration site. The purpose of this study is to functionally characterize the attB in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, and to investigate the presence of Tn5253 attB site into bacterial genomes. Results Analysis of representative Tn5253-carryng transconjugants obtained in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, namely Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes, and Enterococcus faecalis showed that: (i) Tn5253 integrates in rbgA of S. pneumoniae and in orthologous rbgA genes of other bacterial species, (ii) integration occurs always downstream of a 11-bp sequence conserved among streptococcal and enterococcal hosts, (iii) length of the attB site corresponds to length of the duplication after Tn5253 integration, (iv) attB duplication restores rbgA CDS, (v) Tn5253 produced circular forms containing the attTn site at a concentration ranging between 2.0 × 10−5 to 1.2 × 10−2 copies per chromosome depending on bacterial species and strain, (vi) reconstitution of attB sites occurred at 3.7 × 10−5 to 1.7 × 10−2 copies per chromosome. A database search of complete microbial genomes using Tn5253 attB as a probe showed that (i) thirteen attB variants were present in the 85 complete pneumococcal genomes, (ii) in 75 pneumococcal genomes (88.3 %), the attB site was 83 or 84 nucleotides in length, while in 10 (11.7 %) it was 41 nucleotides, (iii) in other 19 bacterial species attB was located in orthologous rbgA genes and its size ranged between 17 and 84 nucleotides, (iv) the 11-bp sequence, which correspond to the last 11 nucleotides of attB sites, is conserved among the different bacterial species and can be considered the core of the Tn5253 integration site. Conclusions A functional characterization of the Tn5253 attB integration site combined with genome analysis contributed to elucidating the potential of Tn5253 horizontal gene transfer among different bacterial species.

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Towards functional characterization of archaeal genomic dark matter

Biochemical Society Transactions ◽

10.1042/bst20180560 ◽

2019 ◽

Vol 47 (1) ◽

pp. 389-398 ◽

Cited By ~ 9

Author(s):

Kira S. Makarova ◽

Yuri I. Wolf ◽

Eugene V. Koonin

Keyword(s):

Dark Matter ◽

Functional Characterization ◽

Comparative Genomic ◽

Hypothetical Proteins ◽

Experimental Characterization ◽

Functional Prediction ◽

Bacterial Genomes ◽

Substantial Fraction ◽

Quantitative Characteristics

Abstract A substantial fraction of archaeal genes, from ∼30% to as much as 80%, encode ‘hypothetical' proteins or genomic ‘dark matter'. Archaeal genomes typically contain a higher fraction of dark matter compared with bacterial genomes, primarily, because isolation and cultivation of most archaea in the laboratory, and accordingly, experimental characterization of archaeal genes, are difficult. In the present study, we present quantitative characteristics of the archaeal genomic dark matter and discuss comparative genomic approaches for functional prediction for ‘hypothetical' proteins. We propose a list of top priority candidates for experimental characterization with a broad distribution among archaea and those that are characteristic of poorly studied major archaeal groups such as Thaumarchaea, DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaeota) and Asgard.

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Cloning of the 5′ regulatory regions and functional characterization of the core promoters of ovine PLAU (u-PA) and SERPIN1 (PAI-1)

Gene ◽

10.1016/j.gene.2011.08.020 ◽

2011 ◽

Vol 489 (1) ◽

pp. 11-20 ◽

Cited By ~ 1

Author(s):

A.D. Lampidonis ◽

G. Theodorou ◽

C. Pecorini ◽

R. Rebucci ◽

A. Baldi ◽

...

Keyword(s):

Functional Characterization ◽

Regulatory Regions ◽

Core Promoters ◽

The Core ◽

Pai 1

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Streptococcus pneumoniae: a Plethora of Temperate Bacteriophages With a Role in Host Genome Rearrangement

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.775402 ◽

2021 ◽

Vol 11 ◽

Author(s):

Antonio J. Martín-Galiano ◽

Ernesto García

Keyword(s):

Streptococcus Pneumoniae ◽

Pneumococcal Disease ◽

Genome Rearrangement ◽

Integration Site ◽

Evolutionary Relationships ◽

Biological Entity ◽

Bacterial Genomes ◽

Gene Encoding ◽

Complete Dataset ◽

Independent Mechanism

Bacteriophages (phages) are viruses that infect bacteria. They are the most abundant biological entity on Earth (current estimates suggest there to be perhaps 1031 particles) and are found nearly everywhere. Temperate phages can integrate into the chromosome of their host, and prophages have been found in abundance in sequenced bacterial genomes. Prophages may modulate the virulence of their host in different ways, e.g., by the secretion of phage-encoded toxins or by mediating bacterial infectivity. Some 70% of Streptococcus pneumoniae (the pneumococcus)—a frequent cause of otitis media, pneumonia, bacteremia and meningitis—isolates harbor one or more prophages. In the present study, over 4000 S. pneumoniae genomes were examined for the presence of prophages, and nearly 90% were found to contain at least one prophage, either defective (47%) or present in full (43%). More than 7000 complete putative integrases, either of the tyrosine (6243) or serine (957) families, and 1210 full-sized endolysins (among them 1180 enzymes corresponding to 318 amino acid-long N-acetylmuramoyl-L-alanine amidases [LytAPPH]) were found. Based on their integration site, 26 different pneumococcal prophage groups were documented. Prophages coding for tRNAs, putative virulence factors and different methyltransferases were also detected. The members of one group of diverse prophages (PPH090) were found to integrate into the 3’ end of the host lytASpn gene encoding the major S. pneumoniae autolysin without disrupting it. The great similarity of the lytASpnand lytAPPH genes (85–92% identity) allowed them to recombine, via an apparent integrase-independent mechanism, to produce different DNA rearrangements within the pneumococcal chromosome. This study provides a complete dataset that can be used to further analyze pneumococcal prophages, their evolutionary relationships, and their role in the pathogenesis of pneumococcal disease.

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Functional Characterization of MigA and WapR: Putative Rhamnosyltransferases Involved in Outer Core Oligosaccharide Biosynthesis of Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.01546-07 ◽

2008 ◽

Vol 190 (6) ◽

pp. 1857-1865 ◽

Cited By ~ 32

Author(s):

Karen K. H. Poon ◽

Erin L. Westman ◽

Evgeny Vinogradov ◽

Shouguang Jin ◽

Joseph S. Lam

Keyword(s):

Pseudomonas Aeruginosa ◽

Functional Characterization ◽

Outer Core ◽

Resonance Spectroscopy ◽

Core Oligosaccharide ◽

Strain Pao1 ◽

The Core ◽

O Antigen ◽

Knockout Mutants

ABSTRACT Pseudomonas aeruginosa lipopolysaccharide (LPS) contains two glycoforms of core oligosaccharide (OS); one form is capped with O antigen through an α-1,3-linked l-rhamnose (l-Rha), while the other is uncapped and contains an α-1,6-linked l-Rha. Two genes in strain PAO1, wapR (PA5000) and migA (PA0705), encode putative glycosyltransferases associated with core biosynthesis. We propose that WapR and MigA are the rhamnosyltransferases responsible for the two linkages of l-Rha to the core. Knockout mutants with mutations in both genes were generated. The wapR mutant produced LPS lacking O antigen, and addition of wapR in trans complemented this defect. The migA mutant produced LPS with a truncated outer core and showed no reactivity to outer core-specific monoclonal antibody (MAb) 5C101. Complementation of this mutant with migA restored reactivity of the LPS to MAb 5C101. Interestingly, LPS from the complemented migA strain was not reactive to MAb 18-19 (specific for the core-plus-one O repeat). This was due to overexpression of MigA in the complemented strain that caused an increase in the proportion of the uncapped core OS, thereby decreasing the amount of the core-plus-one O repeat, indicating that MigA has a regulatory role. The structures of LPS from both mutants were elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry. The capped core of the wapR mutant was found to be truncated and lacked α-1,3-l-Rha. In contrast, uncapped core OS from the migA mutant lacked α-1,6-l-Rha. These results provide evidence that WapR is the α-1,3-rhamnosyltransferase, while MigA is the α-1,6-rhamnosyltransferase.

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Molecular and functional characterization of the interaction of L-ficolin with Streptococcus pneumoniae

Immunobiology ◽

10.1016/j.imbio.2012.08.171 ◽

2012 ◽

Vol 217 (11) ◽

pp. 1188-1189 ◽

Cited By ~ 2

Author(s):

Emilie Stermann ◽

Monique Lacroix ◽

Evelyne Gout ◽

Emmanuelle Laffly ◽

Christian M. Pedersen ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Functional Characterization

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Functional characterization of a cellulose synthase, CtCESA1, from the marine red alga Calliarthron tuberculosum (Corallinales)

Journal of Experimental Botany ◽

10.1093/jxb/erab414 ◽

2021 ◽

Author(s):

Jan Xue ◽

Pallinti Purushotham ◽

Justin F Acheson ◽

Ruoya Ho ◽

Jochen Zimmer ◽

...

Keyword(s):

Cell Wall ◽

Bacterial Species ◽

Functional Characterization ◽

Cellulose Synthase ◽

Red Alga ◽

Land Plant ◽

Primary Cell Wall ◽

Vitro Assay ◽

Red Algal

Abstract In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthase enzymes (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum (Ct) using sequence similarity-based approaches and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESAs. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared to plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal cellulose synthases. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are likely functional differences between the algal and land plant CESAs.

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Functional Characterization of WaaL, a Ligase Associated with Linking O-Antigen Polysaccharide to the Core of Pseudomonas aeruginosa Lipopolysaccharide

Journal of Bacteriology ◽

10.1128/jb.187.9.3002-3012.2005 ◽

2005 ◽

Vol 187 (9) ◽

pp. 3002-3012 ◽

Cited By ~ 84

Author(s):

Priyanka D. Abeyrathne ◽

Craig Daniels ◽

Karen K. H. Poon ◽

Mauricia J. Matewish ◽

Joseph S. Lam

Keyword(s):

Pseudomonas Aeruginosa ◽

Lipid A ◽

Functional Characterization ◽

Wild Type ◽

The Core ◽

O Antigen ◽

Repeat Units ◽

Cytoplasmic Face ◽

O Antigens

ABSTRACT The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of d-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.

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AsnB Mediates Amidation of Meso-Diaminopimelic Acid Residues in the Peptidoglycan of Listeria monocytogenes and Affects Bacterial Surface Properties and Host Cell Invasion

Frontiers in Microbiology ◽

10.3389/fmicb.2021.760253 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lei Sun ◽

Gil Rogiers ◽

Pascal Courtin ◽

Marie-Pierre Chapot-Chartier ◽

Hélène Bierne ◽

...

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Cell Surface ◽

Bacterial Species ◽

Functional Characterization ◽

Diaminopimelic Acid ◽

Bacterial Surface ◽

Gram Positive Bacteria ◽

First Time

A mutant of Listeria monocytogenes ScottA with a transposon in the 5' untranslated region of the asnB gene was identified to be hypersensitive to the antimicrobial t-cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of Listeria is annotated as a glutamine-dependent asparagine synthase, sequence alignment showed that this protein is closely related to a subset of homologs that catalyze the amidation of meso-diaminopimelic acid (mDAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an asnB mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates mDAP amidation in L. monocytogenes. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB mutant, including formation of shorter but thicker cells, susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. In addition, the mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. Analysis by immunofluorescence microscopy revealed that asnB inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to mDAP via its LPXTG motif. Together, this work shows that AsnB of L. monocytogenes, like several of its homologs in related Gram-positive bacteria, mediates the amidation of mDAP residues in the peptidoglycan and, in this way, affects several cell wall and cell surface-related properties. It also for the first time implicates the amidation of peptidoglycan mDAP residues in cell wall anchoring of InlA and in bacterial virulence.

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