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Role of the Anti-Sigma Factor SpoIIAB in Regulation of  G
during Bacillus subtilis Sporulation.Mónica Serrano, 2004.RNA polymerase sigma factor F
initiates the prespore-specific program of gene expression during
Bacillus subtilis sporulation .
F
governs transcription of spoIIIG, encoding the late prespore-specific
regulator
G .
However, transcription of spoIIIG is delayed relative to other
genes under the control of
F,
and after synthesis,
G
is initially kept in an inactive form . Activation of
G
requires the complete engulfment of the prespore by the mother cell
and expression of the spoIIIA and spoIIIJ loci . We
screened for random mutations in spoIIIG that bypassed the
requirement for spoIIIA for the activation of
G .
We found a mutation (spoIIIGE156K) that resulted in an amino
acid substitution at position 156, which is adjacent to the position
of a mutation (E155K) previously shown to prevent interaction of
SpoIIAB with
G .
Comparative modelling techniques and in vivo studies suggested that
the spoIIIGE156K mutation interferes with the interaction of
SpoIIAB with
G .
The
GE156K
isoform restored
G-directed
gene expression to spoIIIA mutant cells . However, expression
of sspE-lacZ in the spoIIIA spoIIIGE156K double
mutant was delayed relative to completion of the engulfment process
and was not confined to the prespore . Rather, ß-galactosidase
accumulated throughout the entire cell at late times in development .
This suggests that the activity of
GE156K
is still regulated in the prespore of a spoIIIA mutant, but
not by SpoIIAB . In agreement with this suggestion, we also found that
expression of spoIIIGE156K from the promoter for the early
prespore-specific gene spoIIQ still resulted in sspE-lacZ
induction at the normal time during sporulation, coincidently with
completion of the engulfment process . In contrast, transcription of
spoIIIGE156K, but not of the wild-type spoIIIG gene,
from the mother cell-specific spoIID promoter permitted the
rapid induction of sspE-lacZ expression . Together, the results
suggest that SpoIIAB is either redundant or has no role in the
regulation of
G
in the prespore .
icaR Encodes a Transcriptional Repressor Involved in Environmental Regulation of ica Operon Expression and Biofilm Formation in Staphylococcus epidermidis. Kevin M. Conlon, 2002.Biofilm formation in Staphylococcus epidermidis is dependent upon the ica operon-encoded polysaccharide intercellular adhesin, which is subject to phase-variable and environmental regulation . The icaR gene, located adjacent to the ica operon, appears to be a member of the tetR family of transcriptional regulators . In the reference strain RP62A, reversible inactivation of the ica operon by IS256 accounts for 25 to 33% of phase variants . In this study, icaA and icaR regulation were compared in RP62A and a biofilm-forming clinical isolate, CSF41498, in which IS256 is absent . Predictably, ica operon expression was detected only in wild-type CSF41498 and RP62A but not in non-IS256-generated phase variants . In contrast, the icaR gene was not expressed in RP62A phase variants but was expressed in CSF41498 variants . An icaR::Emr insertion mutation in CSF41498 resulted in an at least a 5.8-fold increase in ica operon expression but did not significantly alter regulation of the icaR gene itself . Activation of ica operon transcription by ethanol in CSF41498 was icaR dependent . In contrast, a small but significant induction of ica by NaCl and glucose (NaCl-glucose) was observed in the icaR::Emr mutant . In addition, transcription of the icaR gene itself was not significantly affected by NaCl-glucose but was repressed by ethanol . Expression of the ica operon was induced by ethanol or NaCl-glucose in phase variants of CSF41498 (icaR+) but not in RP62A variants (icaR deficient) . These data indicate that icaR encodes a repressor of ica operon transcription required for ethanol but not NaCl-glucose activation of ica operon expression and biofilm formation . Improved Understanding of the Bacterial Vaginal Microbiota of Women before and after Probiotic Instillation. Jeremy P. Burton, 2003.The vaginal bacterial microbiota of 19 premenopausal women was examined by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of the V2-V3 region of the 16S rRNA gene . Ten of the women were studied further to investigate the effect and persistence of vaginally inserted capsules containing viable lactobacilli . PCR-DGGE indicated that most subjects had a microbiota represented by one to three dominant DNA fragments . Analysis of these fragments revealed that 79% of the women possessed sequences with high levels of similarity to Lactobacillus species sequences . Sequences homologous to Lactobacillus iners sequences were the most common and were detected in 42% of the women tested . Alteration of the vaginal microbiota could be detected by PCR-DGGE in several women after the instillation of lactobacilli . Additionally, randomly amplified polymorphic DNA analysis of lactobacilli isolated from selective media demonstrated that the exogenous strains could be detected for up to 21 days in some subjects . This study demonstrates that non-culture-based techniques, such as PCR-DGGE, are useful adjuncts for studies of the vaginal microbiota . Effect of the Environment on Genotypic Diversity of Actinomyces naeslundii and Streptococcus oralis in the Oral Biofilm. James S. Paddick, 2003.The genotypic diversity of Actinomyces naeslundii genospecies 2 (424 isolates) and Streptococcus oralis (446 isolates) strains isolated from two sound approximal sites in all subjects who were either caries active (seven subjects) or caries free (seven subjects) was investigated by using the repetitive extragenic palindromic PCR . The plaque from the caries-active subjects harbored significantly greater proportions of mutans streptococci and lactobacilli and a smaller proportion of A . naeslundii organisms than the plaque sampled from the caries-free subjects . These data confirmed that the sites of the two groups of subjects were subjected to different environmental stresses, probably determined by the prevailing or fluctuating acidic pH values . We tested the hypothesis that the microfloras of the sites subjected to greater stresses (the plaque samples from the caries-active subjects) would exhibit reduced genotypic diversity since the sites would be less favorable . We found that the diversity of A . naeslundii strains did not change (  2 = 0.68; P = 0.41) although the proportional representation of A . naeslundii was significantly reduced (P < 0.05) . Conversely, the diversity of the S . oralis strains increased (2 = 11.71; P = 0.0006) and the proportional representation of S . oralis did not change . We propose that under these environmental conditions the diversity and number of niches within the oral biofilm that could be exploited by S . oralis increased, resulting in the increased genotypic diversity of this species . Apparently, A . naeslundii was not able to exploit the new niches since the prevailing conditions within the niches may have been deleterious and not supportive of its proliferation . These results suggest that environmental stress may modify a biofilm such that the diversity of the niches is increased and that these niches may be successfully exploited by some, but not necessarily all, members of the microbial community .
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