Influence of growth rate and iron limitation on the expression of outer membrane proteins and enterobactin by Klebsiella pneumoniae grown in continuous culture

Abstract

The influence of the growth rate on outer membrane protein composition and enterobactin production was studied with Klebsiella pneumoniae grown under conditions of iron limitation in chemostats. More enterobactin was produced at fast (D = 0.4 h-1) and slow (D = 0.1 h-1) growth rates in continuous cultures than in either logarithmic-or stationary-phase batch cultures. When the growth rate was controlled under conditions of carbon limitation and the iron level was reduced to 0.5 ,M, the iron-regulated outer membrane proteins and enterobactin were induced at the fast growth rate. At the slow growth rate, although the iron-regulated outer membrane proteins were barely visible, a significant level of enterobactin was still produced. These results suggest that under conditions of either carbon or iron limitation, the growth rate can influence the induction of the high-affinity iron uptake system of K. pneumoniae. Other outer membrane proteins, including a 39-kilodalton peptidoglycan-associated protein, were found to vary with the growth rate and nutrient limitation.Iron is an important microbial nutrient which is often not readily available to microorganisms. In aerobic environments at a neutral pH, it predominates as highly insoluble ferric hydroxide polymers (7). During many infections, the withholding of iron by transferrin and lactoferrin constitutes an important component of nonspecific mammalian defense mechanisms (13,17,39,40). The ability to obtain iron is therefore an important virulence factor and, in response to iron deprivation, many microorganisms produce lowmolecular-weight iron-chelating agents (or siderophores) and membrane receptor proteins (17,31,32,39,40). These high-affinity iron uptake systems are expressed by some bacteria growing in vivo during infections in both animals (18, 35) and humans (5,24,36). We recently showed that Klebsiella pneumoniae grown in batch cultures in irondepleted media or in serum induced at least six new ironregulated outer membrane (OM) proteins (IRMPs) together with the iron chelator enterobactin (enterochelin) (41).During infections, bacterial growth rates appear to be slow (4,11,37), and nutrients as well as or other than iron may be growth limiting (6,37,39 siderophore enterobactin by K. pneumoniae. In the carbonlimited chemostat, the medium iron content was lowered to concentrations close to but above the concentration which would be rate limiting. MATERIALS AND METHODSOrganisms and growth conditions. K. pneumoniae DL1, an encapsulated clinical isolate (41), was grown aerobically in continuous cultures at 37°C in glass chemostats at a working volume of 50 ml maintained at a dilution rate of either D = 0.1 h'-(doubling time, 6.9 h) or D = 0.4 h'-(doubling time, 1.7 h). For each experiment, the chemostat was allowed to equilibrate by passing five complete changes of medium through the vessel. Bacteria were also grown to the stationary phase in batch cultures. The simple salts medium (SSM) used for all cultures was that described by Williams et al. (41). This medium wa...

Evolution of the ferric enterobactin receptor in gram-negative bacteria

“…In E. coli the ferric enterobactin receptor also acts as the cognate OM receptor for colicins B and D (25,72). Related members of the family Enterobacteriaceae, including Klebsiella, Salmonella, and Shigella spp., both produce and transport enterobactin (15,42,53); the structural gene for the ferric enterobactin receptor pro-* Corresponding author. tein has been cloned from the latter two bacteria (64a, 67a).…”

“…tein has been cloned from the latter two bacteria (64a, 67a). The effects of iron stress have been studied in many gramnegative bacteria, including Escherichia (12,14,36,48,53), Haemophilus (49,50), Klebsiella (33,42), Neisseria (4,58,73), Pasteurella (16,31,55,66), Pseudomonas (60), Proteus (18), Salmonella (17,20,75), Serratia (46), Shigella (57), Vibrio (2,15,37,69), and Yersinia (8,9,61) spp. These bacteria synthesize numerous iron-regulated membrane proteins (IRMP) whose biochemical functions are for the most part uncharacterized.…”

Effects of growth rate and nutrient limitation on virulence factor production in Burkholderia cepacia

“…1 demonstrate siderophore production in B. cepacia to be growth rate as well as oxygen level dependent. A similar dependence of siderophore production on growth rate has been reported for Klebsiella pneumoniae (21) where under various levels of iron availability in continuous culture, siderophore levels increased with growth rate. At low rates of growth, the iron requirement of B. cepacia may be relatively low such that a basal level may provide sufficient iron for the cells.…”

“…Such changes may result in low growth rates which subsequently affect virulence factor production. These changes have been mimicked in vitro through the use of iron-restricted batch culture (21,25,39). However, in vivo, nutrients as well as or other than iron may be growth limiting (4,32).…”

In vivo monitoring of therapeutic efficacy and virulence profile by bioluminescent Klebsiella pneumoniae.

“…Such mechanisms involve the production of iron chelators known as siderophores that compete with host proteins such as transferrin and hemoglobin over iron by developing higher affinity for binding ferric iron (Fe 3+ ) [158]. K. pneumoniae encodes for four types of siderophores including enterobactin, aerobactin, yersiniabactin, and salmochelin which sequester iron for the bacteria [114,[159][160][161][162][163][164][165][166]. However, only the production of aerobactin and yersiniabactin could be linked with bacterial virulence in murine infection models [162,[167][168][169].…”