Scanning the Escherichia coli chromosome by random transposon mutagenesis and multiple phenotypic screening

Serina, Stefania; Nozza, Francesca; Nicastro, G.; Faggioni, Federico; Mottl, Harald; Dehò, Gianni; Polissi, Alessandra

doi:10.1016/j.resmic.2004.05.006

Cited by 30 publications

(30 citation statements)

References 44 publications

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“…Due to their similarity to ABC transporters (YddA) and TonBdependent outer membrane receptors (YddB), it has been hypothesized that these two proteins may form part of a new iron uptake system (35). Interestingly, the genes were also identified in a transposon mutagenesis screen and found to be required for optimal growth at 37°C in rich medium (57). They are two of the most highly expressed genes at 37°C compared to 23°C (4.8-and 8.0-fold, respectively).…”

Section: Resultsmentioning

confidence: 99%

Human Body Temperature (37°C) Increases the Expression of Iron, Carbohydrate, and Amino Acid Utilization Genes in Escherichia coli K-12

Malhowski

Young

2007

J Bacteriol

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Using DNA microarrays, we identified 126 genes in Escherichia coli K-12 whose expression is increased at human body temperature (37°C) compared to growth at 23°C. Genes involved in the uptake and utilization of amino acids, carbohydrates, and iron dominated the list, supporting a model in which temperature serves as a host cue to increase expression of bacterial genes needed for growth. Using quantitative real-time PCR, we investigated the thermoregulatory response for representative genes in each of these three categories (hisJ, cysP, srlE, garP, fes, and cirA), along with the fimbrial gene papB. Increased expression at 37°C compared to 23°C was retained in both exponential and stationary phases for all of the genes and in most of the various media tested, supporting the relative importance of this cue in adapting to changing environments. Because iron acquisition is important for both growth and virulence, we analyzed the regulation of the iron utilization genes cirA and fes and found that growth in iron-depleted medium abrogated the thermoregulatory effect, with high-level expression at both temperatures, contrasting with papB thermoregulation, which was not greatly altered by limiting iron levels. A positive role for the environmental regulator H-NS was found for fes, cirA, hisJ, and srlE transcription, whereas it had a primarily negative effect on cysP and garP expression. Together, these studies indicate that temperature is a broadly used cue for regulating gene expression in E. coli and that H-NS regulates iron, carbohydrate, and amino acid utilization gene expression.

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Section: Resultsmentioning

confidence: 99%

Human Body Temperature (37°C) Increases the Expression of Iron, Carbohydrate, and Amino Acid Utilization Genes in Escherichia coli K-12

Malhowski

Young

2007

J Bacteriol

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“…5), thus implicating LptB in the control of LptC-LptA interactions. The tight genetic association of lptCAB, which belong to the same yrbG operon (24), and the presence of two ancillary promoters within lptC for the nested lptAB operon (4, 35) may be instrumental in maintaining balanced expression levels of the three encoded proteins.…”

Section: Discussionmentioning

confidence: 99%

“…In nonpermissive (no arabinose) conditions, expression of lptAB might be driven by these minor promoters only, whereas, in permissive conditions, the strong promoter araBp is also active. The viability of ST-190 in the presence of arabinose (24) indicates that, under such conditions, LptC190N is functional, whereas the nonviability of the mutant in nonpermissive condition suggests that lptAB expression is either lacking or insufficient. Here, we show (Fig.…”

Section: Fig 2 Complementation Test Of Lptc Depletion Mutants With DImentioning

confidence: 99%

Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

et al. 2016

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The assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made in Escherichia coli of seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N-and C-terminal regions, and its role in LPS export is not completely understood. Here, we show that the expression level of lptB is a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carries Pa-LptC, the highly divergent LptC orthologue from Pseudomonas aeruginosa. We found that LptB overexpression stabilizes C-terminally truncated LptC mutant proteins, thereby allowing the formation of a sufficient amount of stable IM complexes to support growth. Moreover, the LptB level seems also critical for the assembly of IM complexes carrying Pa-LptC which is otherwise defective in interactions with the E. coli LptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery. IMPORTANCEThe asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens difficult to treat. In Escherichia coli the LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a transenvelope complex. Here, we show that increased expression of the membrane-associated ABC protein LptB can suppress defects of LptC, which participates in the formation of the periplasmic bridge. This reveals functional interactions between these two components and supports a role of LptB in the assembly of the Lpt machine.

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“…Random insertional mutagenesis of P. putida KT2442 was carried out by using the mini-Tn5araC-P BAD transposon, as described elsewhere (Serina et al, 2004).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the last step of the aerobic phenylacetic acid degradation pathway

et al. 2007

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Scanning the Escherichia coli chromosome by random transposon mutagenesis and multiple phenotypic screening

Cited by 30 publications

References 44 publications

Human Body Temperature (37°C) Increases the Expression of Iron, Carbohydrate, and Amino Acid Utilization Genes in Escherichia coli K-12

Human Body Temperature (37°C) Increases the Expression of Iron, Carbohydrate, and Amino Acid Utilization Genes in Escherichia coli K-12

Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

Characterization of the last step of the aerobic phenylacetic acid degradation pathway

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