Allaoui, 1993 (Characterization of the Shigella flexneri ipgD and ipgF genes, which are located in the proximal part of the mxi locus.) [Article]

What is the predicted size and structure of the IpgD protein?

"The ipgD gene is predicted to encode a 538-amino-acid-residue polypeptide with a calculated Mr of 59,764." (Allaoui, 1993)

What was observed regarding IpgD secretion in wild-type vs mutant strains?

"Comparison of the protein contents in the culture supernatants of the wild-type and ipgD strains indicated that the protein of 58 kDa was missing in the culture supernatant of the ipgD mutant" (Allaoui, 1993)

How is ipgD expression regulated by temperature?

"β-Galactosidase activity was 30-fold higher after growth of SF134 at 37°C than at 30°C (640 U versus 25 U), confirming that expression of ipgD, like the invasive phenotype of S. flexneri, is regulated by temperature." (Allaoui, 1993)

What is the genetic organization of ipgD and surrounding genes?

"There are 9 bp between the ipgD stop codon and the ipgE start codon and a 1-bp overlap between the ipgE stop codon and the ipgF start codon. This organization suggests that ipgD, ipgE, and ipgF belong to the same transcriptional unit." (Allaoui, 1993)

What was the effect of ipgD mutation on Shigella virulence?

"The ipgD mutant showed the same ability as the wild-type strain to invade HeLa cells, to induce the formation of plaques on confluent monolayers of HeLa cells, and to provoke keratoconjunctivitis in guinea pigs." (Allaoui, 1993)

How was secretion of IpgD protein confirmed in complementation studies?

"Secretion of the 58-kDa protein was restored in the ipgD mutant harboring plasmid pAB17" (Allaoui, 1993)

What is surprising about IpgD's role in relation to virulence?

"It thus appears that two secreted proteins, IpaA and possibly IpgD, are not involved in the virulent phenotype of S. flexneri. This is very surprising, especially considering the positions of the ipaA and ipgD genes in the invasion region of the virulence plasmid and the presence of the encoded proteins among the few proteins that are secreted into the culture supernatant." (Allaoui, 1993)

Figure 1

Structures of plasmids carrying the ipgD, ipgE, and ipgF genes. The relative positions and orientations of some of the genes identified in the five regions of the virulence plasmid fragment that is necessary for invasion are indicated by arrows on the first line. Below is shown a schematic restriction map of the region encompassing the icsB, ipgD, ipgE, and ipgF genes

Figure 2

Nucleotide sequence of the ipgD, ipgE, and ipgF genes. The nucleotide sequence of the 2,880-bp fragment located upstream from icsB is shown along with the deduced amino acid sequences of IpgD, IpgE, and IpgF and of the N-terminal portion of IcsB.

Figure 3

Analysis of the secreted proteins in the ipgD mutant. Concentrated culture supernatants of S. flexneri BS176 (lane 1), M90T-Sm (lane 2), and SF701 (ipgD) (lane 3) were separated by SDS-PAGE and stained with Coomassie brilliant blue.

Figure 4

Immunoblot analysis of the IpgF-PhoA hybrid proteins. Extracts of E. coli CC118 (ΔphoA) carrying each of several plasmids that express an ipgF-phoA fusion were separated by SDS-PAGE, transferred onto nitrocellulose, and reacted with a serum directed against alkaline phosphatase.

Figure 5

Sequence comparison of IpgF and ORF169. The complete sequence of the S. flexneri IpgF protein (this work) has been aligned with the sequence of ORF169

What bacterial strains were used in the study and how were they grown?

S. flexneri M9OT (wild type), its plasmidless derivative BS176, and M9OT-Sm, a streptomycin-resistant derivative of M9OT, have been previously described. Escherichia coli DH5a was used for plasmid constructions, E. coli SM0lpir was used to transfer plasmids to S. flexneri, and E. coli CC118 was used for TnphoA mutagenesis. Bacteria were grown in Luria-Bertani medium or tryptic soy broth.

How were protein extracts prepared and analyzed?

Bacteria in exponential growth phase were harvested by centrifugation; after filtration through 0.22-µm pore-size filters, proteins present in the culture supernatant were precipitated by the addition of 1/10 (vol/vol) trichloroacetic acid... Electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed

How was the ipgD-lacZ transcriptional fusion constructed?

Plasmid pLAC3 was constructed by cloning a 1.4-kb SspI fragment, extending up to bp 840 in Fig. 2, into the SmaI site located upstream from the lacZ reporter gene in pLAC1. pLAC3 was then transferred by conjugation into S. flexneri M9OT-Sm, with transconjugants selected on plates that contained streptomycin and ampicillin.

How were β-Galactosidase and alkaline phosphatase activities measured?

β-Galactosidase activity was assayed by using the substrate o-nitrophenyl-β-D-galactoside as described by Miller; alkaline phosphatase activity was assayed by using the substrate p-nitrophenyl phosphate as described by Manoil and Beckwith. Both activities are expressed in milli-optical density units at 420 nm per minute per optical density unit at 600 nm