The length of a tetranucleotide repeat tract in Haemophilus influenzae determines the phase variation rate of a gene with homology to type III DNA methyltransferases

2000 ◽  
Vol 35 (1) ◽  
pp. 211-222 ◽  
Author(s):  
Xavier De Bolle ◽  
Christopher D. Bayliss ◽  
Dawn Field ◽  
Tamsin van de Ven ◽  
Nigel J. Saunders ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Phase Variation ◽  
Dna Methyltransferases ◽  
Tetranucleotide Repeat ◽  
Type Iii ◽  
Repeat Tract ◽  
Variation Rate

scholarly journals The role of Dam methylation in phase variation of Haemophilus influenzae genes involved in defence against phage infection

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3361-3369 ◽  
Author(s):  
Piotr Zaleski ◽  
Marek Wojciechowski ◽  
Andrzej Piekarowicz
Keyword(s):  
Haemophilus Influenzae ◽  
Phase Variation ◽  
Phage Infection ◽  
Phase Variable ◽  
Repeat Tract ◽  
Insertional Inactivation ◽  
Phage Dna ◽  
Dam Methylation ◽  
Tetranucleotide Repeats

Haemophilus influenzae uses phase variation (PV) to modulate the activity of its defence systems against phage infection. The PV of the restriction–modification (R-M) system HindI, the main defence system against phage infection and incoming chromosomal and phage DNA in H. influenzae Rd, is driven by changes of the pentanucleotide repeat tract within the coding sequence of the hsdM gene and is influenced by lack of Dam methylation. Phase-variable resistance/sensitivity to phage infection correlates with changes in lipooligosaccharide (LOS) structure and occurs by slippage of tetranucleotide repeats within the gene lic2A, coding for a step in the biosynthesis of LOS. The lack of Dam activity destabilizes the tetranuclotide (5′-CAAT) repeat tract and increases the frequency of switching from sensitivity to resistance to phage infection more than in the opposite direction. The PV of the lgtC gene does not influence resistance or sensitivity to phage infection. Insertional inactivation of lic2A, but not lgtC or lgtF, leads to resistance to phage infection and to the same structure of the LOS as observed among phase-variable phage-resistant variants. This indicates that in the H. influenzae Rd LOS only the first two sugars (Glc-Gal) extending from the third heptose are part of bacterial phage receptors.

Induction of the SOS regulon of Haemophilus influenzae does not affect phase variation rates at tetranucleotide or dinucleotide repeats

Microbiology ◽  
2005 ◽  
Vol 151 (8) ◽  
pp. 2751-2763 ◽  
Author(s):  
Wendy A. Sweetman ◽  
E. Richard Moxon ◽  
Christopher D. Bayliss
Keyword(s):  
Haemophilus Influenzae ◽  
Uv Irradiation ◽  
Phase Variation ◽  
Sos Response ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Dinucleotide Repeats ◽  
Tetranucleotide Repeat ◽  
E Coli ◽  
Lexa Binding

Haemophilus influenzae has microsatellite repeat tracts in 5′ coding regions or promoters of several genes that are important for commensal and virulence behaviour. Changes in repeat number lead to switches in expression of these genes, a process referred to as phase variation. Hence, the virulence behaviour of this organism may be influenced by factors that alter the frequency of mutations in these repeat tracts. In Escherichia coli, induction of the SOS response destabilizes dinucleotide repeat tracts. H. influenzae encodes a homologue of the E. coli SOS repressor, LexA. The H. influenzae genome sequence was screened for the presence of the minimal consensus LexA-binding sequence from E. coli, CTG(N)10CAG, in order to identify genes with the potential to be SOS regulated. Twenty-five genes were identified that had LexA-binding sequences within 200 bp of the start codon. An H. influenzae non-inducible LexA mutant (lexA NI) was generated by site-directed mutagenesis. This mutant showed increased sensitivity, compared with wild-type (WT) cells, to both UV irradiation and mitomycin C (mitC) treatment. Semi-quantitative RT-PCR studies confirmed that H. influenzae mounts a LexA-regulated SOS response following DNA assault. Transcript levels of lexA, recA, recN, recX, ruvA and impA were increased in WT cells following DNA damage but not in lexA NI cells. Induction of the H. influenzae SOS response by UV irradiation or mitC treatment did not lead to any observable SOS-dependent changes in phase variation rates at either dinucleotide or tetranucleotide repeat tracts. Treatment with mitC caused a small increase in phase variation rates in both repeat tracts, independently of an SOS response. We suggest that the difference between H. influenzae and E. coli with regard to the effect of the SOS response on dinucleotide phase variation rates is due to the absence of any of the known trans-lesion synthesis DNA polymerases in H. influenzae.

scholarly journals Identification of the Functional Initiation Codons of a Phase-Variable Gene of Haemophilus influenzae, lic2A, with the Potential for Differential Expression

2006 ◽  
Vol 189 (2) ◽  
pp. 511-521 ◽  
Author(s):  
Kevin Dixon ◽  
Christopher D. Bayliss ◽  
Katherine Makepeace ◽  
E. Richard Moxon ◽  
Derek W. Hood
Keyword(s):  
Gene Expression ◽  
Haemophilus Influenzae ◽  
Phase Variation ◽  
Site Directed Mutagenesis ◽  
Phase Variable ◽  
Tetranucleotide Repeat ◽  
Strong Expression ◽  
Variable Gene ◽  
Weak Expression ◽  
Reading Frames

ABSTRACT Simple sequence repeats located within reading frames mediate phase-variable ON/OFF switches in gene expression by generating frameshifts. Multiple translation initiation codons in different reading frames are found upstream of most Haemophilus influenzae tetranucleotide repeat tracts, raising the possibility of multiple active reading frames and more than two levels of gene expression for these loci. Phase variation between three levels of gene expression (strong, weak, and none) was observed when lic2A was fused to a lacZ reporter gene. The lic2A 5′ CAAT repeat tract is preceded by four 5′ ATG codons (x, y, z1, and z2) in two reading frames. Each of these initiation codons was inactivated by site-directed mutagenesis. Strong expression from frame 1 was associated with x but not y. Weak expression from frame 2 was mainly dependent on the z2 codon, and there was no expression from frame 3. Using monoclonal antibodies specific for a digalactoside epitope of lipopolysaccharide whose synthesis requires Lic2A, two levels (strong and undetectable) of antibody reactivity were detected, suggesting that weak expression of lic2A is not discernible at the phenotypic level. Inactivation of the x initiation codon resulted in loss of strong expression of the digalactoside epitope and elevated killing by human serum. The failure to detect more than two phenotypes for lic2A, despite clear evidence of weak expression from the z1/z2 initiation codons, leaves open the question of whether or not multiple initiation codons are associated with more complex patterns of phenotypic variation rather than classical phase-variable switching between two phenotypes.

scholarly journals Elucidation of the Monoclonal Antibody 5G8-Reactive, Virulence-Associated Lipopolysaccharide Epitope of Haemophilus influenzae and Its Role in Bacterial Resistance to Complement-Mediated Killing

2005 ◽  
Vol 73 (4) ◽  
pp. 2213-2221 ◽  
Author(s):  
Ruth Griffin ◽  
Andrew D. Cox ◽  
Katherine Makepeace ◽  
James C. Richards ◽  
E. Richard Moxon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Haemophilus Influenzae ◽  
Bacterial Resistance ◽  
Inner Core ◽  
Phase Variation ◽  
Phase Variable ◽  
Type B ◽  
Variable Expression ◽  
Virulent Type ◽  
Unknown Structure

ABSTRACT The phase-variable locus lex2 is required for expression of a Haemophilus influenzae lipopolysaccharide (LPS) epitope of previously unknown structure. This epitope, which is reactive with monoclonal antibody (MAb) 5G8, has been associated with virulence of type b strains. When strain RM118 (from the same source as strain Rd), in which the lex2 locus and MAb 5G8 reactivity are absent, was transformed with lex2 DNA, transformants that were reactive with MAb 5G8 were obtained. Surprisingly, the 5G8 reactivity of these transformants was phase variable, although the lex2 locus lacked tetrameric repeats and was constitutively expressed. This phase variation was shown to be the result of phase-variable expression of phosphorylcholine (PCho) such that MAb 5G8 reacted only in the absence of PCho. Structural analysis showed that, compared to RM118, the lex2 transformant had acquired a tetrasaccharide, Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β1,4, linked to the proximal heptose (HepI). A terminal GalNAc was detected in a minority of glycoforms. LPS derived from a mutant of RM7004, a virulent type b strain which naturally expresses lex2 and has LPS containing the same tetrasaccharide linked to HepI as the sole oligosaccharide extension from the inner core, confirmed that GalNAc is not a part of the MAb 5G8-reactive epitope. Thus, MAb 5G8 specifically binds to the structure Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β attached via a 1,4 linkage to HepI of H. influenzae LPS, and we show that the ability to synthesize this novel tetrasaccharide was associated with enhanced bacterial resistance to complement-mediated killing.

scholarly journals Transcriptional Phase Variation of a Type III Restriction-Modification System in Helicobacter pylori

2002 ◽  
Vol 184 (23) ◽  
pp. 6615-6623 ◽  
Author(s):  
Nicolette de Vries ◽  
Dirk Duinsbergen ◽  
Ernst J. Kuipers ◽  
Raymond G. J. Pot ◽  
Patricia Wiesenekker ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Phase Variation ◽  
Transcriptional Level ◽  
Length Variation ◽  
Phase Variable ◽  
Modification System ◽  
Type Iii ◽  
Restriction Modification System ◽  
H Pylori ◽  
Restriction Modification

ABSTRACT Phase variation is important in bacterial pathogenesis, since it generates antigenic variation for the evasion of immune responses and provides a strategy for quick adaptation to environmental changes. In this study, a Helicobacter pylori clone, designated MOD525, was identified that displayed phase-variable lacZ expression. The clone contained a transcriptional lacZ fusion in a putative type III DNA methyltransferase gene (mod, a homolog of the gene JHP1296 of strain J99), organized in an operon-like structure with a putative type III restriction endonuclease gene (res, a homolog of the gene JHP1297), located directly upstream of it. This putative type III restriction-modification system was common in H. pylori, as it was present in 15 out of 16 clinical isolates. Phase variation of the mod gene occurred at the transcriptional level both in clone MOD525 and in the parental H. pylori strain 1061. Further analysis showed that the res gene also displayed transcriptional phase variation and that it was cotranscribed with the mod gene. A homopolymeric cytosine tract (C tract) was present in the 5′ coding region of the res gene. Length variation of this C tract caused the res open reading frame (ORF) to shift in and out of frame, switching the res gene on and off at the translational level. Surprisingly, the presence of an intact res ORF was positively correlated with active transcription of the downstream mod gene. Moreover, the C tract was required for the occurrence of transcriptional phase variation. Our finding that translation and transcription are linked during phase variation through slipped-strand mispairing is new for H. pylori.

Phase variation in a type III restriction-modification system of Helicobacter pylori

2000 ◽  
Vol 118 (4) ◽  
pp. A736 ◽  
Author(s):  
Nicolette Vries de ◽  
Dirk Duinsbergen ◽  
Ernst J. Kuipers ◽  
Patricia Wiesenekker ◽  
Christina M. Vandenbroucke-Grauls ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Phase Variation ◽  
Modification System ◽  
Type Iii ◽  
Restriction Modification System ◽  
Restriction Modification
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