sábado, 20 de marzo de 2010

Contribution of the SirA regulon to biofilm

There are ~2500 serovars of Salmonella enterica with
varying host ranges and disease manifestations. S. enterica
serovar Typhi is the cause of typhoid fever in humans, and
serovar Typhimurium (S. typhimurium) is one of the most
common serovars associated with human gastroenteritis
(Zhang et al., 2003). Within animal hosts, S. typhimurium
actively invades the intestinal epithelial layer preferentially
at the Peyer's patches. Bacteria that penetrate the epithelium
are engulfed by macrophages. Depending on the Salmonella
serovar and the animal species, the bacterium can survive
and replicate within macrophages, and disseminate to the
mesenteric lymph nodes, spleen and liver (Zhang et al.,
2003). Typhoidal salmonellae may survive within their
asymptomatic human hosts by forming biofilms on the
surfaces of gallstones, and this may explain the failure of
antibiotic treatments to clear the pathogen from asympto-
matic hosts (Prouty et al., 2002). Non-typhoidal salmonella
can also colonize gallstones under laboratory conditions
(Prouty et al., 2002), and in addition, they can formbiofilms
on HEp-2 cells that are suspended in a mini-chemostat with
tissue culture medium (Boddicker et al., 2002). Biofilm
Growth conditions and reporter assays. Unless otherwise
stated, bacteria were grown at 37 uC in Luria–Bertani (LB) broth or
on LB agar (1?2% agar, w/v; EM Science). Colonization factor anti-
gen (CFA) medium was prepared with 10 g NZ Amine (Sheffield
Products), 1?5 g yeast extract (USB), 50 mg MgSO4 and 5 mg
MnCl2 per litre of distilled water, pH 7?4 (Suzuki et al., 2002)
When necessary, media were supplemented with 200 mg ampicillin
,50 mg kanamycin ml
,20 mg tetracycline ml
,30 mg chlor-
amphenicol ml
,or40 mg X-Gal ml
. EGTA (Sigma–Aldrich) was
added to a final concentration of 10 mM.
Activity of the b-galactosidase fusions grown in LB broth was assayed
using ONPG as a substrate (Miller, 1972). Maximal effects of SirA are
observed while cells are actively chemotaxing through motility agar
(Goodier & Ahmer, 2001; Teplitski et al., 2003), therefore, the
expression of b-galactosidase fusions was also scored qualitatively in LB
motility agar (0?3%, w/v) supplemented with X-Gal.
Expression of luciferase activity by reporter strains growing on agar
plates or withinmotility agar was visualized by aHamamatsu C2400-32
intensified charge-coupled device (CCD) camera attached to an Argus
20 image processor, or quantified in a Victor-2 multiwell luminometer
The wild-type S. typhimurium formed a biofilm on the
bottom of the well and at the meniscus. The wild-type also
colonized surfaces of the plastic above themeniscus (Fig. 1).
A mutation in sirA decreased biofilm formation by three- to
fivefold (Fig. 2). The sirA mutant formed only a thin ring of
bacterial cells at the meniscus, and no detectable biofilm on
the bottom of the well (Fig. 1). The presence of sirA on a
low-copy-number plasmid complemented the biofilm
defect, while the plasmid vector alone did not (Fig. 3).
Deletion of barA, the gene encoding the sensor kinase for
SirA, also reduced biofilm formation (Fig. 2). Similar to the
sirAmutant, the barAmutant only formed a thin ring of cells

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