INFECTION AND IMMUNITY, June 2008, p. 2520–2530
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 6
Moraxella catarrhalis Expresses an Unusual Hfq Protein?
Ahmed S. Attia,1,3Jennifer L. Sedillo,1Wei Wang,1Wei Liu,1Chad A. Brautigam,2
Wade Winkler,2and Eric J. Hansen1*
Departments of Microbiology1and Biochemistry,2University of Texas Southwestern Medical, Center, Dallas, Texas 75390, and
Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt3
Received 12 December 2007/Returned for modification 8 February 2008/Accepted 12 March 2008
The Hfq protein is recognized as a global regulatory molecule that facilitates certain RNA-RNA interactions
in bacteria. BLAST analysis identified a 630-nucleotide open reading frame in the genome of Moraxella
catarrhalis ATCC 43617 that was highly conserved among M. catarrhalis strains and which encoded a predicted
protein with significant homology to the Hfq protein of Escherichia coli. This protein, containing 210 amino
acids, was more than twice as large as the Hfq proteins previously described for other bacteria. The C-terminal
half of the M. catarrhalis Hfq protein was very hydrophilic and contained two different types of amino acid
repeats. A mutation in the M. catarrhalis hfq gene affected both the growth rate of this organism and its
sensitivity to at least two different types of stress in vitro. Provision of the wild-type M. catarrhalis hfq gene in
trans eliminated these phenotypic differences in the hfq mutant. This M. catarrhalis hfq mutant exhibited altered
expression of some cell envelope proteins relative to the wild-type parent strain and also had a growth
advantage in a continuous flow biofilm system. The presence of the wild-type M. catarrhalis hfq gene in trans
in an E. coli hfq mutant fully reversed the modest growth deficiency of this E. coli mutant and partially reversed
the stress sensitivity of this E. coli mutant to methyl viologen. The use of an electrophoretic mobility shift assay
showed that this M. catarrhalis Hfq protein could bind RNA derived from a gene whose expression was altered
in the M. catarrhalis hfq mutant.
Moraxella catarrhalis is an unencapsulated gram-negative
coccobacillus that was regarded for many years as a harmless
commensal microorganism that could be isolated from the
human nasopharynx. However, within the last few decades, M.
catarrhalis has been shown to be a pathogen that is capable of
producing disease in both adults and children (27, 62). In
infants and very young children, this bacterium is an important
cause of otitis media (27, 40, 62). This organism can also cause
infectious exacerbations of chronic obstructive pulmonary dis-
ease (39, 43, 52) and may be responsible for 2 to 4 million
occurrences of this type each year in the United States (43).
Although there are some data, derived primarily from im-
mune response studies, about which surface antigens of this
organism are expressed during growth in vivo (2, 34, 41, 42),
the molecular bases for expression of most if not all of these
gene products remain to be determined. To date, studies on
gene expression by M. catarrhalis have involved mainly in-
vestigations of phase-variable genes, including uspA1 (28),
uspA2H (65), and hag (37, 45), and genes encoding proteins
involved in restriction and modification (51), and there has
been one recent study which showed that temperature can
affect expression of the UspA1 protein (25). However, with the
exception of the mutant analysis done by Furano and Campag-
nari (18) working with the M. catarrhalis fur gene, no global
regulators of M. catarrhalis have been identified and studied in
detail in this microorganism.
Hfq, or host factor 1, is a global regulatory protein that has
been widely characterized in many bacterial species in recent
years (for reviews, see references 11, 19, and 61). The Hfq
protein forms a hexameric ring-shaped structure similar to that
of the eukaryotic splicing proteins of the Sm family that have
RNA-binding activities (50). Several functions have been as-
cribed to the Hfq protein, starting with its role in the replica-
tion of the RNA phage Q? (17), to facilitating bacterial cell
responses to environmental stresses (for reviews, see refer-
ences 21, 22, 30, and 57). Most of the phenotypes linked to the
Hfq protein were shown to be the result of its ability to func-
tion as an RNA chaperone that allows RNA-RNA interactions
involving mRNA and small RNA molecules (sRNA) (20, 30,
38, 56). These sRNAs typically affect the expression of their
cognate mRNAs by altering either their stability or translation
(58). This fact has allowed identification of additional bacterial
sRNA molecules by their binding to Hfq (68).
We describe here the M. catarrhalis hfq gene and its encoded
protein product, which is more than twice the size of the Hfq
proteins of other bacterial species studied to date. In addition,
an isogenic M. catarrhalis hfq deletion mutant was constructed
and characterized in several in vitro systems to determine
whether the M. catarrhalis Hfq protein was involved in regu-
lating the stress response of this bacterium. Finally, purified
recombinant M. catarrhalis Hfq protein was shown to bind in
vitro to the RNA transcribed from one particular M. catarrhalis
gene. The abundance of transcript from this latter gene was
significantly increased in the M. catarrhalis hfq mutant, making
this gene a possible target for control by the Hfq protein and
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. M. catarrhalis O35E (3)
was used as the wild-type M. catarrhalis strain in the present study. M. catarrhalis
* Corresponding author. Mailing address: Department of Microbi-
ology, University of Texas Southwestern Medical Center, 5323 Harry
Hines Boulevard, Dallas, TX 75390-9048. Phone: (214) 648-5974. Fax:
(214) 648-5905. E-mail: firstname.lastname@example.org.
?Published ahead of print on 24 March 2008.
at UAB LISTER HILL LIBRARY on May 24, 2008
strains were grown in brain heart infusion (BHI) broth (Becton Dickinson,
Sparks, MD) with aeration or on BHI solidified with 1.5% agar at 37°C in an
atmosphere of 95% air and 5% CO2. Autoagglutination of M. catarrhalis strains
was measured as described previously (65). When appropriate, BHI medium was
supplemented with kanamycin (15 ?g/ml) or spectinomycin (15 ?g/ml). Mueller-
Hinton (MH) broth (Becton Dickinson) was used for some experiments as noted.
The Escherichia coli strain MC4100 and its hfq mutant GS081 have been de-
scribed (67). E. coli strains DH5? (48) and TOP10 (Invitrogen, Carlsbad, CA)
were used for cloning purposes. All E. coli strains were grown by using Luria-
Bertani (LB) medium as described previously (48). When necessary, LB medium
was supplemented with ampicillin (100 ?g/ml), kanamycin (30 ?g/ml), or spec-
tinomycin (100 ?g/ml). The M. catarrhalis plasmid cloning vector pWW115 (64)
was used for complementation analysis with M. catarrhalis mutants. Plasmid
pACYC177 was obtained from New England Biolabs (Beverly, MA).
Western blot analysis and protein identification. Whole-cell lysates (WCL)
were prepared as described previously (7). Outer membrane vesicles were pre-
pared from M. catarrhalis by using the method described by Murphy and Loeb
(44). For Western blot analysis, proteins in samples were resolved by using 15%
(wt/vol) polyacrylamide separating gels for sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene diflu-
oride membranes. The Hfq antiserum (described below) was used at a dilution
of 1:1,000 in phosphate-buffered saline–Tween containing 10% (wt/vol) dried
milk and incubated with the membrane either for 2 h at room temperature or
overnight at 4°C. Horseradish peroxidase-conjugated goat anti-mouse antibody
(Jackson Immunoresearch, West Grove, PA) was used as the secondary anti-
body. The antigen-antibody complexes were detected by using Western Light-
ning Chemiluminescence Reagent Plus (New England Nuclear, Boston, MA).
For the identification of proteins that were differentially expressed by the hfq
mutant, proteins in either WCL or outer membrane vesicles were resolved by
SDS-PAGE, transferred to polyvinylidene difluoride, and then stained with Coo-
massie blue. The selected protein bands were excised from the membranes and
subjected to N-terminal amino acid sequence analysis by means of Edman deg-
radation in the Protein Chemistry Technology Center at the University of Texas
Southwestern Medical Center at Dallas.
DNA and RNA isolation. Chromosomal DNA from agar plate-grown M. ca-
tarrhalis cells was isolated by using a DNA Easy kit (Invitrogen). Plasmid DNA
from either M. catarrhalis or E. coli was isolated by using a miniprep spin kit
(Qiagen). RNA was isolated from M. catarrhalis strains grown to early stationary
phase (optical density at 600 nm [OD600] ? 1.5) by using an RNeasy Midi kit
Construction of M. catarrhalis hfq mutants. Overlapping extension PCR (26)
was used to replace most of the hfq-like region of the M. catarrhalis O35E hfq
open reading frame (ORF) with a promoterless kanamycin resistance cartridge
(35). Briefly, with M. catarrhalis O35E chromosomal DNA as the template, the
oligonucleotide primers AA133 (5?-GTAAGCCGTTAAGCCATTGGCAAT-
3?) and AA135 (5?-CCTAGTTAGTCACCCGGTTTGTCCTTTTGACATAAG
TTTCTCCAA-3?) were used to amplify the region (designated amplicon 133-
135) immediately upstream of codon 7 in the hfq ORF, and the primers AA136
A-3?) and AA137 (5?-CGCCCGAATTTGAGATGGCAA-3?) were used to am-
plify the region (designated amplicon 136-137) immediately downstream of
codon 53 in the hfq ORF. The primers AA111 (5?-GGGTGACTAACTAGGA
GGAATAAAT-3?) and AA116 (5?-GGGTCGCATTATTCCCTCCAGGTA-3?)
were used to amplify the promoterless kanamycin resistance (kan) cartridge from
pUC18K3 (35). The primers AA133 and AA116 were used together with am-
plicon 133-135 and the kan cartridge as templates to obtain a PCR product that
contained both the upstream region and the kan cartridge, whereas the primers
AA111 and AA137 were used with amplicon 136-137 and the kan cartridge to
produce a different PCR product that contained the kan cartridge and the
downstream region. These two PCR products were then used together as tem-
plates for another round of PCR using the primers AA133 and AA137. After
verification of the nucleotide sequence of the final PCR product, it was used to
transform wild-type M. catarrhalis O35E. One kanamycin-resistant transformant
was selected for further characterization and was designated O35E.hfq::kan. A
similar approach was used to obtain a PCR product that lacked almost all of the
M. catarrhalis hfq ORF but contained the relevant flanking DNA. The primers
used for this overlapping extension PCR series were the primers AA133 and
AA172 (5?-GGTTTGTCCTTTTGACATAAGTTTCTC-3?) and the primers
AA171 (5?-ATGTCAAAAGG ACAAACCGATTTTGAAAATCAATCCTGA
TTTGGT-3?) and AA137. The final PCR product was used to transform
O35E.hfq::kan and the transformant colonies were screened for the loss of
kanamycin resistance. One kanamycin-sensitive transformant was chosen for
further characterization and was designated O35E?hfq; this mutant contained an
in-frame deletion of almost the entire hfq ORF. Nucleotide sequence analysis
showed two other nucleotide changes near this deletion in this mutant: one in the
intergenic region between the mia and hfq ORFs and the other within the
remainder of the hfq ORF. There were no nucleotide changes in the kpsF gene
located immediately downstream from the hfq gene.
Complementation of the M. catarrhalis hfq mutant. The wild-type M. catar-
rhalis O35E hfq gene was PCR amplified by using the primers AA160 (5?-ATG
GATCCGGCGTCTTCTCATTTACATTGCTT-3?; the BamHI site is under-
lined) and AA159 (5?-TTAGGAGCTCTGAGTTAG AAGGTATCACCC-3?;
the SacI site is underlined) with O35E chromosomal DNA as a template. After
digestion with both BamHI and SacI, the PCR product was ligated with BamHI-
and SacI-digested pWW115 (64), and this ligation mixture was used to transform
O35E. One spectinomycin-resistant transformant was selected for further char-
acterization, and its plasmid was designated pAA200. This plasmid was used to
transform the O35E?hfq mutant to obtain the recombinant strain O35E?hfq
(pAA200). Plasmid pWW115 was used to transform O35E?hfq to obtain a
negative control strain for complementation analysis.
Complementation of an E. coli hfq mutant with the M. catarrhalis Hfq protein.
The primers AA160 and AA293 (5?-AGCTGCAGTGAGTTAGAAGGTATCA
CCC-3?; the PstI site is underlined) were used to amplify the M. catarrhalis hfq
gene using O35E chromosomal DNA as a template. The PCR product was then
digested using BamHI and PstI and ligated to pACYC177 that had been digested
with the same restriction enzymes. The ligation mixture was used to transform
the E. coli hfq mutant GS081 (67). Kanamycin-resistant transformants were
screened for the expression of the M. catarrhalis Hfq protein by Western blot
analysis, and one transformant was selected for further characterization; its
plasmid was designated pAA105. As a negative control, an irrelevant piece of
DNA from the 5? region of the M. catarrhalis O35E uspA2 gene was amplified
using the primers AA16 (5?-CGCGGATCCTGAAAACCATGAAACTT CTCC
C-3?; the BamHI site is underlined) and AA294 (5?-ACCTGCAGTTGGGTAG
CGATTTTGGT GGTGAC CGA-3?; the PstI site is underlined), ligated into the
BamHI/PstI sites of pACYC177, and transformed into E. coli GS081. The re-
sultant control plasmid was designated pAA105B.
Methyl viologen sensitivity assay. Bacterial cells grown overnight on solidified
media were suspended in 5 ml of BHI medium to a final OD600of 0.2. A 500-?l
portion of this suspension was mixed with an equal volume of BHI broth and
then serially diluted. A 10-?l aliquot of each dilution was spotted onto BHI agar
with or without 40 ?M methyl viologen (Sigma, St. Louis, MO). The spots were
allowed to dry, and then the plates were incubated overnight.
Salt sensitivity assay. Bacterial cells were grown, diluted, and spotted as
described immediately above except that the cells were spotted onto BHI agar
plates that contained either the normal concentration of NaCl in BHI (i.e., 1?
NaCl; 5 mg/ml) or 8? NaCl (i.e., 40 mg/ml). The spots were allowed to dry, and
then the plates were incubated for 24 and 48 h, and the growth of the bacteria
was examined at each time point.
Cloning and purification of the M. catarrhalis Hfq protein. The primer pair
AA138 (5?-AGGGATCCGCACAGACAGACAAACCAAC-3?; the BamHI site
is underlined) and AA163 (5?-GT GGTGGTGGTGGTGGTGGGATTGATTT
TCAAAATCGTCATCATA-3?) and the primer pair AA152 (5?-CACCACCAC
CACCACCACTGATTTGGTCATCATCTTGACTTATCC-3?) and AA156 (5?-
CAAAACGCTTAATGTCAGTTT-3?) were used in overlapping extension PCR
to introduce nucleotides encoding six histidine residues (His) at the C terminus
of the M. catarrhalis O35E Hfq protein. The final PCR product was ligated to the
pCR2.1 TOPO vector (Invitrogen) and transformed into E. coli TOP10 cells.
Kanamycin-resistant transformants were screened for His tag expression, and
one transformant was selected for further characterization; its plasmid was des-
ignated pAA201. To purify the His-tagged Hfq protein, recombinant E. coli cells
grown overnight in broth were harvested, suspended in lysis buffer (50 mM
NaH2PO4, 300 mM NaCl, 10 mM imidazole [pH 8.0]) containing lysozyme (1
?g/ml), and incubated on ice for 30 min. The cells were then subjected to
sonication using a Branson Sonifier 450 sonicator (Proquip, Inc., Macedonia,
OH). Unbroken cells and debris were removed by centrifugation at 26,000 ? g
for 20 min at 4°C. The supernatant was then mixed with NiNTA agarose beads
(Qiagen, Valencia, CA) and incubated with agitation at 4°C for 1 h. The sus-
pension was then loaded into a chromatography column and washed four times
(5? bed volume) with washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM
imidazole [pH 8.0]). The His-tagged Hfq protein was eluted using elution buffer
(50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole [pH 8.0]). Selected frac-
tions were pooled, and dialyzed against storage buffer containing 50 mM Tris-
HCl (pH 7.5), 1 mM EDTA, 50 mM NH4Cl, and 20% glycerol (vol/vol). The
concentration of purified, recombinant Hfq protein was determined by using the
Bradford protein assay (Bio-Rad), and then portions of the protein were stored
at ?70°C for later use.
VOL. 76, 2008M. CATARRHALIS Hfq PROTEIN2521
at UAB LISTER HILL LIBRARY on May 24, 2008
Production of polyclonal antibody against the recombinant M. catarrhalis Hfq
protein. The recombinant Hfq protein was used to immunize mice to obtain
polyclonal antibody against this protein. Briefly, 100 ?g of the protein was mixed
with TiterMax (Sigma) and injected intraperitoneally into BALB/c mice. Four
weeks later, the mice were boosted with 30 ?g of the purified protein in Titer-
Max. After two more weeks, the mice were euthanized, and blood was collected
for serum preparation.
RT-PCR analysis. RNA that had been isolated from M. catarrhalis O35E
grown into the early stationary phase of growth was digested with DNase I
(MessageClean kit; GenHunter Corp, Nashville, TN) to remove any DNA con-
tamination. Oligonucleotide primers were designed to amplify the regions be-
tween the hfq gene and the two flanking genes so that each primer would bind to
a sequence within the ORF of each gene. The reverse transcription (RT) reac-
tion was carried out by using MultiScribe reverse transcriptase (Applied Biosys-
tems, Foster City, CA) followed by PCR amplification. As controls, the reaction
was also done using either DNA alone as the template or with RNA template in
the absence of reverse transcriptase.
Real-time RT-PCR. Primers for real-time RT-PCR were designed by using
Primer Express software (Applied Biosystems). The primer pair AA173 (5?-CA
ATGGGCGAAAGCCTGAT-3?) and AA174 (5?-GTGCTTTACAACCAAAA
GGCCT-3?) was specific for 16S RNA, and the primer pair AA305 (5?-TGTTG
AGACGGTGGTTGACAAT-3?) and AA306 (5?-GCCGTACATTTCCTTGGC
AA-3?) was specific for M. catarrhalis ORF 1068 (hereafter referred to as ORF
1068). The reaction was performed as described previously (6). The data analysis
was carried out by using the 7500 System SDS software v.13 (Applied Biosys-
tems), applying the relative quantification ??CTmethod. The level of the ORF
1068 message was normalized according to the level of the 16S RNA, and the
normalized wild-type message level was used as the calibrator. The Student t test
was used for statistical analysis of these data.
Competitive index-based biofilm experiments. The streptomycin-resistant mu-
tant of the wild-type strain O35E (O35E-Smr) (7) and the hfq mutant
O35E.hfq::kan were grown separately in MH broth to a density equivalent to 108
CFU/ml, and then equal volumes of each culture were mixed in a 1:1 ratio. Serial
dilutions of this inoculum mixture were plated on BHI agar plates containing the
appropriate antibiotic to determine the relative percentages of each strain in the
input mixture. A 3-ml portion of the mixture was used to inoculate 200 ml of MH
broth that was then allowed to grow overnight at 37°C with aeration. A second
3-ml portion was used to inoculate a sterile Sorbarod cellulose filter that was
inserted into a short piece of silicone tubing as described previously (45). After
inoculation, sterile MH broth was allowed to drip along the length of the silicone
tubing onto this Sorbarod filter as described previously (45). Cells present in the
resultant biofilm were collected the next day by scraping the silicone tubing,
starting at a point approximately 0.25 in. above the Sorbarod filter and continu-
ing in the direction of the top of the tubing. The harvested cells from the biofilm,
as well as the cells grown in liquid medium, were serially diluted and plated on
agar media containing the appropriate antibiotics to determine the relative
percentage of each strain in the output mixture. These data were subjected to
repeated-measures analysis and pairwise comparisons were performed by using
the Tukey HSD procedure at the 0.05 significance level (SAS 9.13; SAS, Inc.,
Preparation of a radiolabeled RNA probe. The primers AA373 (5?-GTCAA
ATTTCACTATGCATTATCTT-3?) and AA281 (5?-TGACTGTGTCTGTATC
GCC-3?) were used to amplify an ?300-bp fragment that contained the putative
transcriptional start point of ORF 1068 and part of the 5? region of this ORF.
This PCR fragment was used as a template for another PCR using the primers
AA281 and AA374 (5?-CAGAGATGCATAATACGACTCACTATAGGGAG
AGTCAAATTTCACTATGCATTATCTT-3?; the T7 core promoter sequence
is underlined). The resultant PCR product AA373-AA281 was used in an vitro
transcription reaction using a T7 MAXIscript kit (Ambion, Austin, TX) in the
presence of [?-32P]UTP for 1 h at 37°C according to the manufacturer’s protocol.
The DNA template was then digested with Turbo DNase (Ambion), and the
RNA product was partially purified by using an RNeasy MinElute cleanup kit
EMSA. The radiolabeled RNA probe described immediately above was used
in an electrophoretic mobility shift assay (EMSA) with the purified recombinant
M. catarrhalis Hfq protein. Briefly, equivalent amounts of RNA fragment (ca.
20,000 cpm) were incubated with increasing concentrations of purified Hfq
protein in a 25-?l reaction volume in a binding buffer containing 10 mM Tris-
HCl (pH 8.0), 1 mM EDTA, 80 mM NaCl, 10% (vol/vol) glycerol, and 0.01%
(vol/vol) dodecyl maltoside for 30 min at 37°C. Each reaction tube also contained
E. coli tRNA (100 ng/?l; Roche) to minimize nonspecific binding. The reaction
mixtures were then loaded onto a native 6% polyacrylamide gel and run in 0.5?
Tris-borate-EDTA at room temperature at 200 V for 2 h. The gel was then
exposed to a storage phosphor intensifying screen (GE Healthcare, Piscataway,
NJ) and scanned by using a Storm 820 scanner (GE Healthcare). The image was
analyzed by using ImageQuant v.5.2 software (Molecular Dynamics, Sunnyvale,
CA). To determine whether binding was specific for the RNA fragment used in
this experiment, the binding reaction was also carried out in the presence of 25
ng of the same RNA fragment that had not been radiolabeled.
Identification of an Hfq-like protein encoded by M. catar-
rhalis. The use of the nucleotide sequence of the E. coli hfq
gene in a BLAST search against the M. catarrhalis ATCC 43617
genome (GenBank accession numbers AX067426 to AX067466)
identified a Hfq-like protein encoded by a 630-nucleotide (nt)
ORF. The predicted encoded protein contained 210 amino
acids (Fig. 1), and nucleotide sequence analysis of the hfq ORF
from five additional M. catarrhalis strains (O35E, FIN2344,
V1118, 7169, and O12E) revealed that this ORF was identical
among all six strains (data not shown). Amino acid alignment
of the predicted M. catarrhalis Hfq protein with Hfq proteins
from other well-studied bacterial species (Fig. 1) showed high
degrees of identity and similarity between these latter proteins
and the N-terminal one-third of the M. catarrhalis Hfq protein.
For example, all of the amino acids predicted to form the
RNA-binding pocket in different Hfq proteins (5, 49, 50, 69)
are conserved in the M. catarrhalis Hfq protein (Fig. 1).
In contrast to these other Hfq proteins that typically con-
tained between 77 and 99 amino acids, the M. catarrhalis Hfq
protein contained more than 100 additional amino acids; these
were present in the C-terminal half of this macromolecule (Fig.
1). Hydrophilicity analysis of the amino acid sequence of the E.
coli Hfq protein indicated that the C-terminal domain contain-
ing approximately 30 amino acids was hydrophilic (data not
shown). Analysis of the much larger number of additional
amino acids in the C-terminal half of the M. catarrhalis Hfq
protein indicated that this region is highly hydrophilic in nature
but is not predicted to form a regular secondary structure (data
not shown). The amino acid sequence GF appears nine times
in these 142 amino acids in the M. catarrhalis Hfq protein, and
the sequence GFERQTQGGFDRGGMGYQ appears twice.
When the complete amino acid sequence of the M. catarrhalis
Hfq protein was used in a BLAST search against the nonre-
dundant protein databases, the predicted proteins with the
highest level of identity (E values  of 2 ? 10?30) were found
in Psychrobacter species. These three different Psychrobacter pro-
teins (accession numbers ZP_01272067.1, YP_580756.1, and
YP_264208.1) each possessed predicted Hfq proteins that were
very similar in size to the M. catarrhalis Hfq protein, containing
183 or 201 amino acids, but no description of these Psy-
chrobacter proteins has been published to date.
Genetic organization of the M. catarrhalis hfq chromosomal
locus. Upon examination of the hfq chromosomal locus in M.
catarrhalis strain ATCC 43617, we found that the hfq gene was
preceded by an miaA gene which encodes a predicted delta
2-isopentenylpyrophosphate transferase (12) (Fig. 2A). Imme-
diately downstream of the M. catarrhalis hfq gene, we found an
ORF with homology to kpsF, which encodes a predicted arabi-
nose-5-phosphate isomerase (60).
RT-PCR analysis was performed with RNA from M. ca-
tarrhalis O35E to determine whether the M. catarrhalis hfq
gene was transcriptionally linked to either of the two flank-
2522 ATTIA ET AL.INFECT. IMMUN.
at UAB LISTER HILL LIBRARY on May 24, 2008
ing genes. Primer AA135, which binds to the 5? end of the hfq
gene on the negative strand (Fig. 2A), was used in a reverse
transcriptase reaction, followed by a PCR using the primer
AA138, which binds to the 3? end of the miaA gene (Fig. 2A).
This RT-PCR yielded a ?200-bp PCR product (Fig. 2B, lane
4) similar to the one obtained using O35E chromosomal DNA
as a template (Fig. 2B, lane 2), and no product was obtained in
the PCR in which the reverse transcriptase enzyme was not
added (Fig. 2B, lane 3). These results indicated that M. ca-
tarrhalis hfq and miaA genes are linked transcriptionally.
Primer AA134, which binds to the 5? end of the kpsF gene on
the negative strand (Fig. 2A), was used in an RT-PCR with
primer AA169, which binds to the 3? end of the hfq gene (Fig.
2A). The results obtained with these two primers (Fig. 2B,
lanes 5 to 7) indicated that the hfq and kpsF genes are tran-
scriptionally linked in M. catarrhalis O35E.
Construction and complementation of M. catarrhalis hfq mu-
tants. Overlapping extension PCR, followed by transformation
and allelic exchange, was used to construct two different M.
catarrhalis hfq mutants. The first mutant, O35E.hfq::kan, had a
promoterless kan cartridge in place of most of the hfq-like
region (i.e., nt 19 to 159) in the hfq ORF (Fig. 3A). This
mutant was then used to construct the deletion mutant
O35E?hfq in which the region from nt 16 to 612, comprising
almost the entire hfq ORF, was deleted in-frame (Fig. 3B) as
described in Materials and Methods. The M. catarrhalis cloning
vector pWW115 carrying the wild-type hfq gene from M. ca-
tarrhalis O35E was used to complement the O35E?hfq mutant,
yielding the recombinant M. catarrhalis strain O35E?hfq
(pAA200). The recombinant M. catarrhalis strain O35E?hfq
(pWW115) was used as a negative control. When subjected to
Western blot analysis with mouse polyclonal antibody raised
against the recombinant M. catarrhalis Hfq protein, both the
wild-type O35E strain and the complemented O35E?hfq mu-
tant (Fig. 3C, lanes 1 and 3, respectively) expressed an anti-
body-reactive band of approximately 23 kDa, which is very
similar to the predicted mass of the M. catarrhalis Hfq protein.
This same antibody-reactive band was absent from both the hfq
deletion mutant O35E?hfq and the negative control strain
O35E?hfq(pWW115) (Fig. 3C, lanes 2 and 4, respectively).
The M. catarrhalis hfq mutant is growth deficient. A pheno-
typic feature common to the hfq mutants of some other bac-
terial species is a growth deficiency in vitro (15, 53, 59). Upon
examination of the growth characteristics of the M. catarrhalis
hfq deletion mutant, it was observed that this mutant (Fig. 4,
solid squares) exhibited a modest growth deficiency compared
to the wild-type parent strain O35E (Fig. 4, open diamonds).
This deficiency involved both a prolongation of the lag phase
FIG. 1. Comparison of the deduced amino acid sequence of the M. catarrhalis Hfq protein with the sequences of Hfq proteins from other
bacteria. The deduced amino acid sequence of the M. catarrhalis ATCC 43617 Hfq protein (top sequence) was aligned to the amino acid sequences
of seven other bacterial Hfq proteins. The asterisks indicate the amino acids predicted to form the nucleotide-binding pocket in other Hfq proteins
(5, 49, 50, 69). This figure was generated by using the CLUSTAL W alignment program in MacVector (version 6.5).
VOL. 76, 2008 M. CATARRHALIS Hfq PROTEIN 2523
at UAB LISTER HILL LIBRARY on May 24, 2008
and a very slight decrease in final culture density relative to
that obtained with the wild-type parent strain. The hfq mutant
did not autoagglutinate more rapidly than the wild-type parent
strain (data not shown). The observed growth deficiency of the
O35E?hfq mutant was substantially reversed by the introduc-
tion of a wild-type M. catarrhalis hfq gene in trans (Fig. 4, open
triangles). However, for reasons that remain unclear, the neg-
ative control strain O35E?hfq(pWW115) (Fig. 4, solid circles)
showed a greater growth deficiency than that observed with the
hfq deletion mutant.
The M. catarrhalis hfq mutant exhibits increased sensitivity
to stress. Methyl viologen (paraquat) is a redox-cycling agent
that causes cytotoxicity by producing a cytosolic superoxide
flux (23). No difference was apparent in the growth of the
wild-type parent strain O35E and the hfq deletion mutant
when they were spotted onto BHI agar (Fig. 5A), but the hfq
deletion mutant was ?100-fold more sensitive to 40 ?M
methyl viologen than was the wild-type parent strain (Fig. 5B).
The increase in susceptibility to methyl viologen observed with
the O35E?hfq mutant was reversed by complementing this
mutant with a wild-type M. catarrhalis hfq gene provided in
trans, whereas the same mutant containing only the plasmid
vector pWW115 was as sensitive to methyl viologen as the hfq
deletion mutant (Fig. 5B).
Both the wild-type strain O35E and the O35E?hfq mutant
FIG. 3. Construction of M. catarrhalis hfq mutants. (A and B) Sche-
matic representation of the M. catarrhalis chromosomal locus containing
the hfq gene and flanking regions in the hfq mutant O35E.hfq::kan
(A) and the hfq deletion mutant O35E?hfq (B). The relative positions of
the different primers used for PCR are indicated by the arrows. (C) West-
ern blot analysis using mouse polyclonal antibody raised against the re-
combinant M. catarrhalis O35E Hfq protein to probe WCL derived from
2), the O35E?hfq mutant containing the pAA200 plasmid with the wild-
type O35E hfq gene [?hfq(pAA200), lane 3], and this same mutant con-
taining only the plasmid vector [?hfq(pWW115), lane 4].
FIG. 2. Genetic organization of the M. catarrhalis hfq chromosomal locus. (A) Schematic representation of the M. catarrhalis hfq chromosomal
locus in M. catarrhalis ATCC 43617, including the two genes flanking the hfq ORF. The relative positions of the different oligonucleotide primers
used for PCR and RT-PCR are indicated by the arrows. (B) Photograph of an agarose gel showing the results of a RT-PCR experiment involving
the hfq ORF and the two flanking genes. The primer pair used in each experiment is indicated on the top of the gel. Lane 1 contains DNA size
markers. Lanes 2 and 5 contain samples from RT-PCRs where DNA was used as the template. Lanes 3 and 6 contain samples from RT-PCRs
where RNA was used as the template but no reverse transcriptase was added [(?)RT]. Lanes 4 and 7 contain samples from RT-PCRs where RNA
was used as the template and reverse transcriptase was added [(?)RT].
2524ATTIA ET AL.INFECT. IMMUN.
at UAB LISTER HILL LIBRARY on May 24, 2008
were inhibited by a high salt concentration (compare Fig. 6A
and B). However, the O35E?hfq mutant was more sensitive to
inhibition by a high concentration of salt. The difference in
growth of the wild-type strain and the hfq mutant in the pres-
ence of a high salt concentration was modest after 24 h (Fig.
6B), whereas the difference became much more apparent after
48 h (Fig. 6D). In the presence of a high salt concentration, the
complemented mutant O35E?hfq(pAA200) grew to the same
extent as the wild-type parent strain (Fig. 6B and D) and the
negative control strain O35E?hfq(pWW115) grew to lesser
extent than the O35E?hfq mutant (Fig. 6B and 6D).
Effect of the hfq mutation on protein expression by M. ca-
tarrhalis. Comparison of the protein profiles of WCL of the
wild-type O35E parent strain and the hfq deletion mutant (Fig.
7A) revealed a band of ?18 kDa that was more highly ex-
pressed in the mutant. N-terminal sequence analysis of the
protein contained in this band yielded the amino acid sequence
GVFEFAKDIG. When used to search the M. catarrhalis ATCC
43617 genome (GenBank accession numbers AX067426 to
AX067466), this sequence most closely matched that of a pre-
dicted M. catarrhalis protein that contains a LysM peptidogly-
can-binding motif. Use of this protein to search the nonredun-
dant protein databases showed that it was most similar (67%
identity; E value ? 10?51) to a predicted protein containing a
LysM peptidoglycan-binding motif (accession no. YP_579785.1)
FIG. 4. Growth profiles of wild-type, mutant, and complemented
mutant strains of M. catarrhalis. Cells of the wild-type strain O35E
(?), the hfq deletion mutant O35E?hfq (f), the complemented hfq
deletion mutant O35E?hfq(pAA200) (‚), and the negative control
O35E?hfq(pWW115) (F) were suspended in BHI broth to an OD600
of 1.0 and then diluted 1:20. These suspensions were allowed to grow
with aeration at 37°C, and the growth was monitored by measuring the
absorbance at 600 nm every hour.
FIG. 5. Effect of the hfq mutation on the sensitivity of M. catarrhalis
to methyl viologen. Bacterial cells of each strain were suspended to the
same OD600and then serially diluted and spotted onto BHI agar
(A) and BHI agar containing 40 ?M methyl viologen (B). Four strains
were tested: wild-type O35E strain (WT), the O35E?hfq mutant
(?hfq), the O35E?hfq mutant containing the pAA200 plasmid with the
wild-type O35E hfq gene [?hfq(pAA200)], and this same mutant con-
taining only the plasmid vector [?hfq(pWW115)]. The plates were
dried and incubated overnight, and the resultant growth was photo-
FIG. 6. Effect of the hfq mutation on the sensitivity of M. catarrhalis
to increased salt concentration. Bacterial cells of the same four strains
described in Fig. 5 were suspended to the same OD600and then serially
diluted and plated onto BHI agar containing 1? NaCl (5 mg/ml; A and
C) and onto BHI agar containing 8? NaCl (40 mg/ml; B and D) and
incubated for 24 h (A and B) and 48 h (C and D) at 37°C.
VOL. 76, 2008 M. CATARRHALIS Hfq PROTEIN2525
at UAB LISTER HILL LIBRARY on May 24, 2008
from Psychrobacter cryohalolentis. Comparison of the outer
membrane protein profiles of the wild-type strain O35E and
the hfq deletion mutant (Fig. 7B) showed several bands that
appeared to be slightly more abundant in the hfq mutant rel-
ative to the wild-type strain. These proteins, with apparent
sizes of approximately 75, 25, and 18 kDa, were identified by
N-terminal amino acid sequence analysis as the outer mem-
brane proteins CopB (4), OMP G1b (1), and OMP J (24) (data
Effect of the hfq mutation on biofilm development in vitro. A
competitive index approach (14, 29) was used to determine
whether the hfq mutation affected the ability of M. catarrhalis
to form a biofilm in vitro in a continuous flow system. Cells of
the streptomycin-resistant mutant of the O35E parent strain
(i.e., O35E-Smr) were mixed with an equivalent number of
cells of the hfq mutant O35E.hfq::kan. When this mixture was
used to inoculate broth and allowed to grow overnight, the
proportions of the O35E-Smrstrain and the O35E.hfq::kan
mutant recovered after overnight growth were not different
(P ? 0.05) to those in the inoculum (Fig. 8A). These data are
in agreement with the previous comparison of the growth
curves of the wild-type strain and the hfq deletion mutant in
broth, where the extent of growth of the hfq mutant in station-
ary phase was just slightly less than that of the wild-type strain
(Fig. 4). In contrast, when a mixture of O35E-Smrand the
O35E.hfq::kan mutant was used to inoculate a continuous-flow
biofilm system, the hfq mutant clearly predominated (P ? 0.05)
in the population that was recovered from the biofilm after
overnight growth (Fig. 8B).
The M. catarrhalis Hfq protein can complement an E. coli hfq
mutation. The M. catarrhalis Hfq protein is considerably larger
than the Hfq proteins of other bacteria, with the nonhomolo-
gous region in the C-terminal half being larger than the Hfq
proteins of these other organisms. In order to investigate the
possibility that these numerous additional amino acids in the C
terminus of the M. catarrhalis Hfq protein might interfere with
the ability of this protein to function in the same manner as
other Hfq proteins, the wild-type M. catarrhalis hfq gene was
cloned into the E. coli hfq mutant GS081 (67) on plasmid
FIG. 7. Effects of the hfq mutation on protein expression by M. catarrhalis. Proteins present in WCL (A) and outer membrane vesicles (OMV)
(B) from the wild-type strain O35E and the hfq mutant O35E?hfq were resolved by SDS-PAGE and stained with Coomassie blue. The positions
of some of the bands that were more abundant in the hfq mutant are indicated by the black arrows. Proteins contained in the four selected bands
were subjected to N-terminal amino acid sequence analysis. Protein molecular mass position markers (in kilodaltons) are indicated on the left side
of each panel.
FIG. 8. The M. catarrhalis hfq mutant exhibits a growth advantage
in a biofilm system. Cells of M. catarrhalis O35E-Smr(f) and
O35E.hfq::kan (?) were mixed together (input) and used to inoculate
MH broth in a flask (A) and a Sorbarod filter in silicone tubing that
was supplied with a continuous flow of MH broth (B). The cultures
were allowed to grow overnight at 37°C and then harvested (output),
serially diluted, and plated onto agar media containing the appropriate
antibiotics to determine the relative percentages of each strain in the
mixture. The data presented are the means of three independent
2526ATTIA ET AL.INFECT. IMMUN.
at UAB LISTER HILL LIBRARY on May 24, 2008
pAA105. As a negative control, this same E. coli hfq mutant
was transformed with the same plasmid vector that had an
irrelevant DNA insert (i.e., pAA105B).
These two transformants, together with the E. coli parent
strain MC4100 and the E. coli hfq mutant GS081, were each
grown in LB broth to allow comparison of their growth rates
(Fig. 9A). The E. coli hfq mutant GS081 (Fig. 9A, solid
squares) showed a modest growth defect in LB broth relative
to the parent strain (Fig. 9A, open diamonds). Expression of
the M. catarrhalis Hfq protein in this mutant (Fig. 9A, solid
triangles) reversed this growth defect, whereas the hfq mutant
containing the control plasmid pAA105B (Fig. 9A, open cir-
cles) had a growth defect similar to that of the original GS081
hfq mutant. The E. coli hfq mutant GS081 showed increased
susceptibility to 40 ?M methyl viologen relative to the parent
strain MC4100 (Fig. 9C). The expression of the M. catarrhalis
Hfq protein in this E. coli hfq mutant partially reversed the
decrease in the resistance of this mutant to methyl viologen (Fig.
9C), while the negative control strain E. coli GS081(pAA105B)
was as sensitive to methyl viologen as GS081 (Fig. 9C). No
obvious differences in the growth of these same four strains
were apparent when they were spotted on plain BHI agar (Fig.
The M. catarrhalis Hfq protein binds RNA. The hallmark of
previously characterized Hfq proteins is their ability to bind
RNA, acting as an RNA chaperone (20, 30, 38, 56). In order to
test the ability of the M. catarrhalis Hfq protein to bind RNA,
we chose a potential target that might be regulated through
interaction with Hfq. One of the proteins that was highly ex-
pressed in the M. catarrhalis hfq mutant was the macromole-
cule that contained a LysM peptidoglycan-binding motif (Fig.
7A). This protein is encoded by a gene that was annotated as
ORF 1068 in the M. catarrhalis ATCC 43617 genome (66).
Real-time RT-PCR was performed to compare the levels of
mRNA transcribed from this gene in the wild-type strain O35E
and in the O35E?hfq deletion mutant. The level of ORF 1068
transcript in the O35E?hfq cells was at least four times greater
than the amount of this same message in the wild-type strain
(P ? 0.027; Fig. 10A). It could be inferred from these data that
Hfq is affecting the level of detectable mRNA derived from
this particular gene.
In order to determine whether this effect might involve an
interaction between the Hfq protein and the ORF 1068
mRNA, in vitro transcription was used to transcribe a 317-nt
RNA fragment containing 217 nt from the 5? end of this ORF
and 100 nt of the upstream region, which has a putative tran-
scriptional start site. Equal amounts of this RNA probe were
incubated with increasing concentrations of the purified, re-
combinant M. catarrhalis Hfq protein in an EMSA (Fig. 10B).
The Hfq protein was shown to be able to bind this RNA
fragment, as evidenced by the apparent retardation in the
electrophoretic mobility of the radiolabeled RNA probe in the
presence of concentrations of Hfq protein greater than 62.5
nM (Fig. 10B, lanes 5 to 8). The experiment was repeated in
the presence of 25 ng of the same 317-nt RNA fragment that
was not radiolabeled. The presence of the unlabeled RNA
probe resulted in an increase in the Hfq protein concentration
required to produce a change in the electrophoretic mobility of
the radiolabeled probe, from 62.5 to 500 nM (Fig. 10C, lanes 5
The Hfq protein has been characterized in several bacterial
species in recent years (10, 13, 15, 16, 53, 55). It was first
recognized as the E. coli host factor necessary for replication of
RNA phage Q? (17) and subsequently as a RNA chaperone
and global effector molecular that orchestrates mRNA-sRNA
interactions, frequently in response to environmental signals
(for reviews, see references 11, 19, 32, and 61). Initial analysis
of the M. catarrhalis hfq gene showed that this ORF is pre-
dicted to encode an Hfq protein that contained double the
FIG. 9. Complementation of an E. coli hfq mutant with the M.
catarrhalis Hfq protein. (A) Growth curves of wild-type, mutant, and
recombinant E. coli strains. The wild-type strain MC4100 (?), the hfq
mutant GS081 (f), the hfq mutant with the M. catarrhalis hfq gene
provided in trans [GS081(pAA105)] (Œ), and the hfq mutant contain-
ing the negative control plasmid [GS081(pAA105B)] (E) were sus-
pended in LB broth to an OD600of 1.0 and then diluted 1:20. These
cultures were allowed to grow with aeration at 37°C, and the growth
was monitored by measuring the absorbance at 600 nm every hour. (B
and C) Cells of the wild-type, mutant, and recombinant E. coli strains
were suspended to the same OD600and then serially diluted and
spotted onto BHI agar (B) and onto BHI agar containing 40 ?M
methyl viologen (C). The plates were dried and incubated overnight at
VOL. 76, 2008M. CATARRHALIS Hfq PROTEIN 2527
at UAB LISTER HILL LIBRARY on May 24, 2008
number of amino acids present in other Hfq homologues that
have been previously studied (Fig. 1) and that the additional
amino acids are located in the C-terminal half of the protein
and form a highly hydrophilic sequence. Western plot analysis
confirmed that M. catarrhalis expresses an Hfq protein which
exhibits a mass in SDS-PAGE consistent with that predicted
from the deduced amino acid sequence (Fig. 3). Searching the
nonredundant protein databases for homologues of the M.
catarrhalis Hfq protein showed that three Psychrobacter species
have genes encoding an Hfq protein similar in size to that of M.
catarrhalis, but the C-terminal region of these Psychrobacter
proteins had only moderate identity with that of M. catarrhalis
(data not shown). The redundancy and tandem repeat present
in the amino acid sequence of the C-terminal portion of the M.
catarrhalis Hfq protein suggest that this span of the hfq ORF
arose from at least one duplication event. The exact role of this
long, highly hydrophilic region in the C terminus of the M.
catarrhalis Hfq protein is not clear at this time, but it appar-
ently did not eliminate the ability of the M. catarrhalis Hfq
protein to complement an E. coli hfq mutation (Fig. 9).
The ability of the M. catarrhalis Hfq protein to complement
this E. coli hfq mutant suggested that this M. catarrhalis gene
product could interact with RNA and led us to prove directly
that this protein could bind at least one M. catarrhalis RNA
species. Examination of the effect of an hfq mutation on the
expression of proteins by M. catarrhalis revealed that several
proteins detectable in either WCL or outer membranes were
either upregulated or more abundant in the absence of the Hfq
protein (Fig. 7). One of these genes whose mRNA was defi-
nitely more abundant in the hfq mutant, based on real-time
RT-PCR assays (Fig. 10A), was ORF 1068, which encoded an
18-kDa macromolecule that contained a predicted LysM motif.
LysM or lysine motif domains are thought to function as pep-
tidoglycan-binding domains and are found in some enzymes
that degrade this bacterial cell wall constituent (8, 9) but also
in eukaryotic proteins (46).
Although sRNAs have not been described to date in M.
catarrhalis, the observed effect of the hfq mutation on ORF
1068 mRNA levels raised the possibility that an sRNA might
be involved in controlling expression of this gene. Identifica-
tion of a possible sRNA that interacts with ORF 1068 mRNA
was beyond the scope of the present study, but we took advan-
tage of the fact that Hfq has been shown to bind to at least
some mRNA molecules that are targets for sRNA (19, 36).
When we used the 5? end of the ORF 1068 transcript in an
EMSA with purified, recombinant M. catarrhalis Hfq, this pro-
tein bound the RNA (Fig. 10B and C). It can be inferred from
these data that the M. catarrhalis Hfq protein, either alone or
in concert with sRNA components, affects the abundance of
the ORF 1068 transcript.
Phenotypic characterization of the M. catarrhalis hfq mutant
showed that it had similarities to hfq mutants of some other
bacterial species, such as a slight growth deficiency in liquid
medium (33, 54, 59) and increased sensitivity to oxidative and
osmotic stresses (59, 63). The M. catarrhalis hfq mutant had an
interesting and unexpected phenotype in a continuous-flow
biofilm system where, in competitive index experiments, it be-
came the predominant member of the biofilm after overnight
growth in vitro (Fig. 8). To the best of the authors’ knowledge,
the effect of an hfq mutation on biofilm formation in vitro by
other bacteria has not been reported to date, and a ready
explanation for this apparent growth advantage of the M. ca-
tarrhalis hfq mutant is not apparent. However, the modest
increase in expression or relative abundance of several outer
membrane proteins, including CopB (4), OMP G1b (1), and
OMP J (24), in the M. catarrhalis hfq mutant raises the possi-
bility that overall outer membrane architecture may have been
affected in this mutant. If so, then this change in the surface of
the mutant may have had some effect on its ability to form a
biofilm in this model system. Although hfq mutations fre-
quently affect the virulence potential of some bacterial patho-
gens (13, 15, 47, 53, 54), the current lack of an animal model
for M. catarrhalis disease (27, 62) precludes testing the effect of
this mutation on the virulence of M. catarrhalis.
In conclusion, the data presented here indicated that the
mucosal pathogen M. catarrhalis expresses an Hfq protein that
is much larger than the Hfq proteins of other, well-studied
FIG. 10. The M. catarrhalis Hfq protein binds RNA. (A) Real-time
RT-PCR analysis expression of the ORF 1068 gene that encodes
the LysM motif-containing protein. Total RNA isolated from the wild-
type O35E strain (?) and the O35E?hfq mutant (f) was used for
real-time RT-PCR with oligonucleotide primers specific for the ORF
1068 gene and 16S RNA. The data are presented as the fold increase
using the normalized level of the wild-type O35E ORF 1068 mRNA as
the calibrator. These data represent the mean of two independent
experiments (each performed with samples in triplicate), and the error
bars represent the standard deviations. (B and C) EMSA. A radiola-
beled RNA probe containing part of the ORF 1068 mRNA was incu-
bated with increasing concentrations of the purified M. catarrhalis Hfq
protein in the presence of 100 ng of E. coli tRNA/?l (B) or in the
presence of 100 ng of E. coli tRNA/?l and 25 ng of unlabeled RNA
probe (C). The binding reactions were then subjected to electrophore-
sis in a native 6% (wt/vol) polyacrylamide gel. The bands were visual-
ized by exposing the gel to a storage phosphor intensifying screen that
was then scanned by using a phosphorimager.
2528ATTIA ET AL.INFECT. IMMUN.
at UAB LISTER HILL LIBRARY on May 24, 2008
bacteria. This Hfq protein can bind M. catarrhalis mRNA and
likely interacts with sRNAs in a manner similar to that de-
scribed for other Hfq proteins. Identification of sRNAs ex-
pressed by M. catarrhalis will be the subject of future research
This study was supported by U.S. Public Health Service grant no.
AI36344 to E.J.H.
We thank Gisela Storz for helpful comments and for providing E.
coli strains MC4100 and GS081 and both Kaiping Deng and Maria
Labandeira for generous assistance and helpful discussions.
1. Adlowitz, D. G., T. Hiltke, A. J. Lesse, and T. F. Murphy. 2004. Identification
and characterization of outer membrane proteins G1a and G1b of Moraxella
catarrhalis. Vaccine 22:2533–2540.
2. Adlowitz, D. G., C. Kirkham, S. Sethi, and T. F. Murphy. 2006. Human
serum and mucosal antibody responses to outer membrane protein G1b of
Moraxella catarrhalis in chronic obstructive pulmonary disease. FEMS Im-
munol. Med. Microbiol. 46:139–146.
3. Aebi, C., I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas,
G. H. McCracken, Jr., and E. J. Hansen. 1997. A protective epitope of
Moraxella catarrhalis is encoded by two different genes. Infect. Immun. 65:
4. Aebi, C., B. Stone, M. Beucher, L. D. Cope, I. Maciver, S. E. Thomas, G. H.
McCracken, Jr., P. F. Sparling, and E. J. Hansen. 1996. Expression of the
CopB outer membrane protein by Moraxella catarrhalis is regulated by iron
and affects iron acquisition from transferrin and lactoferrin. Infect. Immun.
5. Arluison, V., P. Derreumaux, F. Allemand, M. Folichon, E. Hajnsdorf, and
P. Regnier. 2002. Structural modelling of the Sm-like protein Hfq from
Escherichia coli. J. Mol. Biol. 320:705–712.
6. Attia, A. S., and E. J. Hansen. 2006. A conserved tetranucleotide repeat is
necessary for wild-type expression of the Moraxella catarrhalis UspA2 pro-
tein. J. Bacteriol. 188:7840–7852.
7. Attia, A. S., E. R. Lafontaine, J. L. Latimer, C. Aebi, G. A. Syrogiannopoulos,
and E. J. Hansen. 2005. The UspA2 protein of Moraxella catarrhalis is
directly involved in the expression of serum resistance. Infect. Immun. 73:
8. Bateman, A., and M. Bycroft. 2000. The structure of a LysM domain from
Escherichia coli membrane-bound lytic murein transglycosylase D (MltD). J.
Mol. Biol. 299:1113–1119.
9. Birkeland, N. K. 1994. Cloning, molecular characterization, and expression
of the genes encoding the lytic functions of lactococcal bacteriophage phi
LC3: a dual lysis system of modular design. Can. J. Microbiol. 40:658–665.
10. Bohn, C., C. Rigoulay, and P. Bouloc. 2007. No detectable effect of RNA-
binding protein Hfq absence in Staphylococcus aureus. BMC. Microbiol.
11. Brennan, R. G., and T. M. Link. 2007. Hfq structure, function and ligand
binding. Curr. Opin. Microbiol. 10:125–133.
12. Caillet, J., and L. Droogmans. 1988. Molecular cloning of the Escherichia
coli miaA gene involved in the formation of delta 2-isopentenyl adenosine in
tRNA. J. Bacteriol. 170:4147–4152.
13. Christiansen, J. K., M. H. Larsen, H. Ingmer, L. Sogaard-Andersen, and
B. H. Kallipolitis. 2004. The RNA-binding protein Hfq of Listeria monocy-
togenes: role in stress tolerance and virulence. J. Bacteriol. 186:3355–3362.
14. Darwin, A. J., and V. L. Miller. 1999. Identification of Yersinia enterocolitica
genes affecting survival in an animal host using signature-tagged transposon
mutagenesis. Mol. Microbiol. 32:51–62.
15. Ding, Y., B. M. Davis, and M. K. Waldor. 2004. Hfq is essential for Vibrio
cholerae virulence and downregulates sigma expression. Mol. Microbiol. 53:
16. Figueroa-Bossi, N., S. Lemire, D. Maloriol, R. Balbontin, J. Casadesus, and
L. Bossi. 2006. Loss of Hfq activates the ?E-dependent envelope stress
response in Salmonella enterica. Mol. Microbiol. 62:838–852.
17. Franze de Fernandez, M. T., L. Eoyang, and J. T. August. 1968. Factor
fraction required for the synthesis of bacteriophage Q?-RNA. Nature 219:
18. Furano, K., and A. A. Campagnari. 2003. Inactivation of the Moraxella
catarrhalis 7169 ferric uptake regulator increases susceptibility to the bacte-
ricidal activity of normal human sera. Infect. Immun. 71:1843–1848.
19. Geissmann, T. A., and D. Touati. 2004. Hfq, a new chaperoning role: binding
to messenger RNA determines access for small RNA regulator. EMBO J.
20. Gottesman, S. 2004. The small RNA regulators of Escherichia coli: roles and
mechanisms. Annu. Rev. Microbiol. 58:303–328.
21. Gottesman, S. 2005. Micros for microbes: non-coding regulatory RNAs in
bacteria. Trends Genet. 21:399–404.
22. Guillier, M., S. Gottesman, and G. Storz. 2006. Modulating the outer mem-
brane with small RNAs. Genes Dev. 20:2338–2348.
23. Hassan, H. M., and I. Fridovich. 1978. Superoxide radical and the oxygen
enhancement of the toxicity of paraquat in Escherichia coli. J. Biol. Chem.
24. Hays, J. P., S. van Selm, T. Hoogenboezem, S. Estevao, K. Eadie, P. van
Keelen, J. Tommassen, A. van Belkum, and P. W. Hermans. 2005. Identifi-
cation and characterization of a novel outer membrane protein (OMP J) of
Moraxella catarrhalis that exists in two major forms. J. Bacteriol. 187:7977–
25. Heiniger, N., R. Troller, P. S. Meier, and C. Aebi. 2005. Cold shock response
of the UspA1 outer membrane adhesin of Moraxella catarrhalis. Infect.
26. Horton, R. M., Z. Cai, S. N. Ho, and L. R. Pease. 1990. Gene splicing by
overlap extension: tailor-made genes using the polymerase chain reaction.
27. Karalus, R., and A. Campagnari. 2000. Moraxella catarrhalis: a review of an
important human mucosal pathogen. Microbes. Infect. 2:547–559.
28. Lafontaine, E. R., N. J. Wagner, and E. J. Hansen. 2001. Expression of the
Moraxella catarrhalis UspA1 protein undergoes phase variation and is regu-
lated at the transcriptional level. J. Bacteriol. 183:1540–1551.
29. Lawlor, M. S., J. Hsu, P. D. Rick, and V. L. Miller. 2005. Identification of
Klebsiella pneumoniae virulence determinants using an intranasal infection
model. Mol. Microbiol. 58:1054–1073.
30. Majdalani, N., C. K. Vanderpool, and S. Gottesman. 2005. Bacterial small
RNA regulators. Crit. Rev. Biochem. Mol. Biol. 40:93–113.
31. Marchler-Bauer, A., and S. H. Bryant. 2004. CD-Search: protein domain
annotations on the fly. Nucleic Acids Res. 32:W327–W331.
32. Masse, E., H. Salvail, G. Desnoyers, and M. Arguin. 2007. Small RNAs
controlling iron metabolism. Curr. Opin. Microbiol. 10:140–145.
33. McNealy, T. L., V. Forsbach-Birk, C. Shi, and R. Marre. 2005. The Hfq
homolog in Legionella pneumophila demonstrates regulation by LetA and
RpoS and interacts with the global regulator CsrA. J. Bacteriol. 187:1527–
34. Meier, P. S., S. Freiburghaus, A. Martin, N. Heiniger, R. Troller, and C.
Aebi. 2003. Mucosal immune response to specific outer membrane proteins
of Moraxella catarrhalis in young children. Pediatr. Infect. Dis. J. 22:256–262.
35. Menard, R., P. J. Sansonetti, and C. Parsot. 1993. Nonpolar mutagenesis of
the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri
entry into epithelial cells. J. Bacteriol. 175:5899–5906.
36. Moll, I., D. Leitsch, T. Steinhauser, and U. Blasi. 2003. RNA chaperone
activity of the Sm-like Hfq protein. EMBO Rep. 4:284–289.
37. Mollenkvist, A., T. Nordstrom, C. Hallden, J. J. Christensen, A. Forsgren,
and K. Riesbeck. 2003. The Moraxella catarrhalis immunoglobulin D-binding
protein MID has conserved sequences and is regulated by a mechanism
corresponding to phase variation. J. Bacteriol. 185:2285–2295.
38. Moller, T., T. Franch, P. Hojrup, D. R. Keene, H. P. Bachinger, R. G.
Brennan, and P. Valentin-Hansen. 2002. Hfq: a bacterial Sm-like protein
that mediates RNA-RNA interaction. Mol. Cell 9:23–30.
39. Murphy, T. F. 1996. Branhamella catarrhalis: epidemiology, surface antigenic
structure, and immune response. Microbiol. Rev. 60:267.
40. Murphy, T. F. 2000. Bacterial otitis media: pathogenetic considerations.
Pediatr. Infect. Dis. J. 19:S9–S15.
41. Murphy, T. F., A. L. Brauer, C. Aebi, and S. Sethi. 2005. Antigenic specificity
of the mucosal antibody response to Moraxella catarrhalis in chronic obstruc-
tive pulmonary disease. Infect. Immun. 73:8161–8166.
42. Murphy, T. F., A. L. Brauer, C. Aebi, and S. Sethi. 2005. Identification of
surface antigens of Moraxella catarrhalis as targets of human serum antibody
responses in chronic obstructive pulmonary disease. Infect. Immun. 73:3471–
43. Murphy, T. F., A. L. Brauer, B. J. Grant, and S. Sethi. 2005. Moraxella
catarrhalis in chronic obstructive pulmonary disease: burden of disease and
immune response. Am. J. Respir. Crit. Care Med. 172:195–199.
44. Murphy, T. F., and M. R. Loeb. 1989. Isolation of the outer membrane of
Branhamella catarrhalis. Microb. Pathog. 6:159–174.
45. Pearson, M. M., E. R. Lafontaine, N. J. Wagner, J. W. St.Geme, III, and E. J.
Hansen. 2002. A hag mutant of Moraxella catarrhalis strain O35E is deficient
in hemagglutination, autoagglutination, and immunoglobulin D-binding ac-
tivities. Infect. Immun. 70:4523–4533.
46. Ponting, C. P., L. Aravind, J. Schultz, P. Bork, and E. V. Koonin. 1999.
Eukaryotic signalling domain homologues in archaea and bacteria: ancient
ancestry and horizontal gene transfer. J. Mol. Biol. 289:729–745.
47. Robertson, G. T., and R. M. Roop, Jr. 1999. The Brucella abortus host factor
I (HF-I) protein contributes to stress resistance during stationary phase and
is a major determinant of virulence in mice. Mol. Microbiol. 34:690–700.
48. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.
49. Sauter, C., J. Basquin, and D. Suck. 2003. Sm-like proteins in Eubacteria: the
crystal structure of the Hfq protein from Escherichia coli. Nucleic Acids Res.
50. Schumacher, M. A., R. F. Pearson, T. Moller, P. Valentin-Hansen, and R. G.
VOL. 76, 2008 M. CATARRHALIS Hfq PROTEIN2529
at UAB LISTER HILL LIBRARY on May 24, 2008
Brennan. 2002. Structures of the pleiotropic translational regulator Hfq and Download full-text
an Hfq-RNA complex: a bacterial Sm-like protein. EMBO J. 21:3546–3556.
51. Seib, K. L., I. R. A. Peak, and M. P. Jennings. 2002. Phase variable restric-
tion-modification systems in Moraxella catarrhalis. FEMS Immunol. Med.
52. Sethi, S., N. Evans, B. J. Grant, and T. F. Murphy. 2002. New strains of
bacteria and exacerbations of chronic obstructive pulmonary disease.
N. Engl. J. Med. 347:465–471.
53. Sittka, A., V. Pfeiffer, K. Tedin, and J. Vogel. 2007. The RNA chaperone Hfq
is essential for the virulence of Salmonella typhimurium. Mol. Microbiol.
54. Sonnleitner, E., S. Hagens, F. Rosenau, S. Wilhelm, A. Habel, K. E. Jager,
and U. Blasi. 2003. Reduced virulence of a hfq mutant of Pseudomonas
aeruginosa O1. Microb. Pathog. 35:217–228.
55. Sonnleitner, E., M. Schuster, T. Sorger-Domenigg, E. P. Greenberg, and U.
Blasi. 2006. Hfq-dependent alterations of the transcriptome profile and
effects on quorum sensing in Pseudomonas aeruginosa. Mol. Microbiol. 59:
56. Storz, G., S. Altuvia, and K. M. Wassarman. 2005. An abundance of RNA
regulators. Annu. Rev. Biochem. 74:199–217.
57. Storz, G., and D. Haas. 2007. A guide to small RNAs in microorganisms.
Curr. Opin. Microbiol. 10:93–95.
58. Tjaden, B., S. S. Goodwin, J. A. Opdyke, M. Guillier, D. X. Fu, S. Gottesman,
and G. Storz. 2006. Target prediction for small, noncoding RNAs in bacteria.
Nucleic Acids Res. 34:2791–2802.
59. Tsui, H. C., H. C. Leung, and M. E. Winkler. 1994. Characterization of
broadly pleiotropic phenotypes caused by an hfq insertion mutation in Esch-
erichia coli K-12. Mol. Microbiol. 13:35–49.
60. Tzeng, Y. L., A. Datta, C. Strole, V. S. Kolli, M. R. Birck, W. P. Taylor, R. W.
Carlson, R. W. Woodard, and D. S. Stephens. 2002. KpsF is the arabinose-
5-phosphate isomerase required for 3-deoxy-D-manno-octulosonic acid bio-
synthesis and for both lipooligosaccharide assembly and capsular polysac-
charide expression in Neisseria meningitidis. J. Biol. Chem. 277:24103–24113.
61. Valentin-Hansen, P., M. Eriksen, and C. Udesen. 2004. The bacterial Sm-
like protein Hfq: a key player in RNA transactions. Mol. Microbiol. 51:1525–
62. Verduin, C. M., C. Hol, A. Fleer, H. van Dijk, and A. Van Belkum. 2002.
Moraxella catarrhalis: from emerging to established pathogen. Clin. Micro-
biol. Rev. 15:125–144.
63. Wachi, M., A. Takada, and K. Nagai. 1999. Overproduction of the outer-
membrane proteins FepA and FhuE responsible for iron transport in Esch-
erichia coli hfq::cat mutant. Biochem. Biophys. Res. Commun. 264:525–529.
64. Wang, W., and E. J. Hansen. 2006. Plasmid pWW115, a cloning vector for
use with Moraxella catarrhalis. Plasmid 56:133–137.
65. Wang, W., M. M. Pearson, A. S. Attia, R. J. Blick, and E. J. Hansen. 2007.
A UspA2H-negative variant of Moraxella catarrhalis strain O46E has a de-
letion in a homopolymeric nucleotide repeat common to uspA2H genes.
Infect. Immun. 75:2035–2045.
66. Wang, W., L. Reitzer, D. A. Rasko, M. M. Pearson, R. J. Blick, C. Laurence,
and E. J. Hansen. 2007. Metabolic analysis of Moraxella catarrhalis and the
effect of selected in vitro growth conditions on global gene expression. Infect.
67. Zhang, A., K. M. Wassarman, J. Ortega, A. C. Steven, and G. Storz. 2002.
The Sm-like Hfq protein increases OxyS RNA interaction with target
mRNAs. Mol. Cell 9:11–22.
68. Zhang, A., K. M. Wassarman, C. Rosenow, B. C. Tjaden, G. Storz, and S.
Gottesman. 2003. Global analysis of small RNA and mRNA targets of Hfq.
Mol. Microbiol. 50:1111–1124.
69. Ziolkowska, K., P. Derreumaux, M. Folichon, O. Pellegrini, P. Regnier, I. V.
Boni, and E. Hajnsdorf. 2006. Hfq variant with altered RNA binding func-
tions. Nucleic Acids Res. 34:709–720.
Editor: J. L. Flynn
2530 ATTIA ET AL.INFECT. IMMUN.
at UAB LISTER HILL LIBRARY on May 24, 2008