Mutational analysis of potential CRE0364, 0937 and 1181 targets

1
Characterization of three new
competence-regulated operons in Haemophilus
influenzae T.M. VanWagoner, P.W. Whitby, T.W.
Seale, D.J. Morton and T.L. Stull Department of
Pediatrics, University of Oklahoma Health Science
Center, Oklahoma City, OK 73104
Mutational analysis of potential CRE0364, 0937
and 1181 targets The comparative genomic
analysis indicated that the original assignments
for CRE0364, CRE0937 and CRE1181 may have been
incorrect. Furthermore, Q-PCR analysis confirmed
that transcription of HI0366, HI0938 and HI0939
and HI1182/1183 (ligA) all increased in response
to competence-inducing conditions while the
originally proposed CRE targets were not
upregulated. In order to determine whether these
new members of the competence regulon were
actually involved in competence and
transformation, insertion or deletion mutations
were created in HI0365, HI0366, HI0939 and ligA.
These mutant strains were then tested to assess
their competence and transformation ability
(Table 2). The HI0365 mutant was
as proficient as the wild-type in the ability to
uptake DNA and be transformed to novobiocin
resistance. In contrast, the HI0366 mutant strain
was unable to be transformed or to uptake DNA,
yet had only a moderate decrease in DNA binding
proficiency. The lack of polar effects in the
HI0365 mutant was later shown to be the result of
constitutive expression of HI0366 from the
insertion marker (data not shown). The HI0939
mutant was completely unable to be transformed
and was severely impacted in the ability to bind
and uptake DNA. Transformation in the ligA mutant
was only moderately impacted (5-fold decrease)
and the mutant was proficient in both DNA binding
and uptake. Further in silico analysis The
products of several of the genes shown in this
study to be upregulated by competence induction
exhibit similarities to proteins known to be
involved with the type II secreton (T2S) and type
IV pili (Tfp), components of which are integral
parts of transformation systems in other
bacteria3. Homologs of other members of these
systems have been identified in H. influenzae and
have demonstrable roles in competence and
transformation in this species. The product of
HI0366 shares weak homology (29 identity) to
PilF of Pseudomonas aeruginosa in this organism,
pilF mutants are unable to form discernable Tfp.
The pilF locus is also extremely similar to the
HI0366 locus (Fig 6). In both organisms, pilF and
HI0366 appear to be the only non-expendable gene
in the locus involved in pilus or transformation
activities. Furthermore, BLAST searches against
the conserved domain database (rpsblast)
identified matches of PulG for HI0938 and PulJ
for HI0939. These proteins are type IV
pseudopilins that form part of the T2S system,
which has been best described for the secretion
of pullulanase in Klebsiella oxytoca. The other
pullulanase operon homologs located in H.
influenzae are all situated in CRE-controlled
operons, and mutations in these operons result in
the abolition of DNA binding and uptake.
Summary In conclusion, we have
demonstrated the ability of comparative genomics
to identify new competence operons. As the
amount of genomic data continues to increase,
such in silico approaches will be a valuable tool
to assist in discovering new members of
regulatory networks. We also utilized Q-PCR to
assess transcriptional profiles of individual
genes. Comparison of the transcription of genes
contiguous with the three examined CRE sites
indicated regulation consistent with that of
other identified competence-regulated genes. The
ability to examine gene expression using Q-PCR
eliminates the need for lacZ fusions and is a
powerful method for limited gene expression
studies. Using this combined in silico,
transcriptional and mutational analysis, we
identified three new members of the competence
regulon in H. influenzae and further demonstrated
the importance of the type II secreton for
transformation in H. influenzae. 1 VanWagoner
et al. (Oct 2004) J Bacteriology.
1866409-6421. 2 Macfadyen, L.P. (2000) J. Theor.
Biol. 207349-359. 3 Chen, I. and Dubnau D.
(2004) Nat. Rev. Microbiol. 2241-249.
Q-PCR analysis of gene expression during
competence induction
Abstract Haemophilus influenzae is one of a
growing number of bacteria in which the natural
ability to uptake exogenous DNA for potential
genomic transformation has been recognized. To
date, several operons involved in transformation
in this organism have been described. These
operons are characterized by a conserved 22-bp
regulatory element upstream of the first gene and
are induced coincident with transfer from rich to
nutrient-depleted media. The previously
identified operons are comprised of genes
encoding proteins that include members of the
type II secretion system and type IV pili, shown
to be essential for transformation in other
bacteria, and with other proteins previously
identified as required for transformation in H.
influenzae. In the current study, three novel
competence operons were identified by comparative
genomics and transcriptional analysis. These
operons have been further characterized by
construction of null mutants and examination of
the resulting transformation phenotypes. The
putative protein encoded by HI0366 was shown to
be essential for DNA uptake, but not binding, and
is homologous to a protein shown to be required
for pilus biogenesis and twitching motility in
Pseudomonas aeruginosa. An insertion in HI0939
abolished both DNA binding and uptake. The
predicted product of this gene shares
characteristics with PulJ, a pseudopilin involved
in pullulanase export in Klebsiella oxytoca.
Introduction Haemophilus influenzae is
the etiological agent of several human diseases
including meningitis, otitis media and
respiratory infections in patients with
predisposing conditions including cystic fibrosis
and chronic obstructive pulmonary disease (COPD).
This species is among a large number of
organisms, including other members of the family
Pasteurellaceae, that are capable of
internalizing exogenous DNA for incorporation
into the bacterial chromosome, a process termed
transformation. Transformation of H. influenzae
has been observed in vivo in diffusion chambers
implanted in the peritoneal cavity in rats, and a
recent study reported horizontal transfer of
ompP2 gene sequences between H. influenzae
strains colonizing the respiratory tract of a
patient with COPD. Most of the operons
containing genes known to be directly involved in
DNA uptake and transformation in H. influenzae
have a conserved 22-bp palindromic element called
the competence regulatory element (CRE) directly
upstream of the transcriptional start sites. The
product of HI0601 tfoX (sxy), is required for
transcription of operons containing the CRE and
insertions into tfoX completely abolish
transformation development. Ten putative CRE
sites have been previously identified in the Rd
KW20 genome (Figure 1).2 Mutational analysis of
genes in five of these operons has confirmed a
role of those genes in transformation in H.
influenzae. While many of the CRE sites are
located between divergently transcribed operons,
divergent transcription from a CRE site has not
been described. We applied in silico,
transcriptional and mutational analysis to
characterize three of the previously unexamined
CRE regions (CRE0364, CRE0937 and CRE1181) to
determine their role in transformation in H.
influenzae.
Figure 5- Transcriptional analysis using
Quantitative, real-time PCR (Q-PCR) was employed
to assess whether transcription of genes
contiguous to the putative CRE elements increased
in response to competence-inducing conditions
(MIV). Transcription profiles of these genes were
compared to profiles of the putative competence
regulatory gene tfoX, and the known
competence-regulated genes rec-2 and comA. Values
represent the fold change in expression with
respect to levels a control (pretreatment) sample
as determined by the 2(-??Ct) method. The
transformation frequency at each time point is
indicated by the closed circles
Figure 2- Organization and conservation of the
CRE0364 region in the Pasteurellaceae. Black
arrows indicate genes whose location in reference
to the CRE element is conserved across the
family. Grey arrows indicate genes with partial
positional conservation. Homologs of HI0365 and
HI0366 are always associated with a putative CRE
site. HI0364, originally proposed to be the
target of CRE0364, was not associated with a
discernable CRE sequence.
Figure 3- Organization and conservation of the
CRE0937 region in the Pasteurellaceae. Black
arrows indicate genes whose location in reference
to the CRE element is conserved across the
family. Grey arrows indicate genes with partial
positional conservation. Homologs of HI0938,
HI0939, HI0941 and recC are always co-associated
with a putative CRE site. HI0937, originally
proposed to be the target of CRE0937, was not
associated with a discernable CRE sequence.
Figure 1- Putative CRE regions in H. influenzae
Rd KW20 Organization of ORFs contiguous to the
putative CRE elements. Genes shown in black
indicate biological data confirming their role in
transformation in H. influenzae. Genes shown in
grey indicate mutations in the operon affect
transformation but polar effects prevent
individual characterization.
Figure 6- The H. influenzae HI0366 and P.
aeruginosa pilF loci. Numbers correspond to the
HI gene designation in the Rd KW20 genome
annotation. P. aeruginosa numbers correspond to
the HI of the closest homolog in the H.
influenzae genome.
Comparative genomic analysis of the CRE loci in
the Pasteurellaceae In order to gain
insight into whether CRE0364, CRE0937 and CRE1182
were competence-associated and which
genes/operons were the likely regulatory target
of the element, a comparative genomic approach
was utilized. Homologs of each of the ORFs
downstream of the CRE elements were located in
the genomic sequences of the Pasteurella members
listed in Table 1.
Figure 4- Organization and conservation of the
CRE01181 region in the Pasteurellaceae. Black
arrows indicate genes whose location in reference
to the CRE element is conserved across the
family. Grey arrows indicate genes with partial
positional conservation. HI1182 and HI1183
actually form a single coding sequence encoding
an ATP-dependent DNA ligase. HI1182/1183 homologs
were not found in all of the Pasteurellaceae, but
when found, were associated with a putative CRE
element. HI1181 (gmhA), originally proposed to be
the target of CRE1181, was not associated with a
discernable CRE sequence.