TnphoA: a transposon probe for protein export signals.

Manoil, Colin; Beckwith, Jon

doi:10.1073/pnas.82.23.8129

Cited by 975 publications

(815 citation statements)

References 31 publications

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“…4). In contrast, the alkaline phosphatase activity of Pkn9-PhoA-2, containing the hydrophobic stretch, was 3923 U, which was similar to the 4919 U of activity found in the strain harbouring pCH2 (Manoil and Beckwith, 1985) (Fig. 4).…”

Section: Pkn9 Contains a Transmembrane Domainsupporting

confidence: 73%

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

Hanlon

Inouye

1997

Molecular Microbiology

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SummaryThe Myxococcus xanthus gene, pkn9, encodes a protein that contains significant homology with eukaryotic Ser/ Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus. This region also shows 29, 25 and 29% identity with myosin light-chain kinase, protein kinase C, and cAMP-dependent protein kinase, respectively. A 22-residue hydrophobic transmembrane domain separates the kinase domain from the 173-residue C-terminal domain that resides on the outside of the inner membrane. The C-terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I-III by the insertion of a kanamycin-resistance gene resulted in slightly delayed, smaller and more-crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Twodimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9-1-4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting-body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.

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Section: Pkn9 Contains a Transmembrane Domainsupporting

confidence: 73%

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

Hanlon

Inouye

1997

Molecular Microbiology

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“…In order to generate insertional mutations in potential membrane-protein genes, S. flexneri strain SA514(pMA9), a chloramphenicol-resistant (Cm R ) derivative of SA100 that retained invasion and intercellular spread abilities, was mated with SM10 pir (pRT733), which carries TnphoA (Manoil and Beckwith, 1985) on a suicide vector (Taylor et al, 1989). Kanamycin-resistant (Km R ), Cm R colonies were selected and then screened for PhoA þ and for their ability to bind Congo red dye (Crb þ ), a characteristic of invasive Shigella strains (Payne and Finkelstein, 1977).…”

Section: Tnphoa Mutagenesismentioning

confidence: 99%

Effect of mutations in Shigella flexneri chromosomal and plasmid‐encoded lipopolysaccharide genes on invasion and serum resistance

Hong

Payne

1997

Molecular Microbiology

117

120

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SummaryThis study shows that both length and distribution of lipopolysaccharide (LPS) are important for Shigella flexneri invasion and virulence. Mutants were generated in the chromosomal LPS synthesis genes rfa, rfb, and rol, and in a plasmid-encoded O-antigen chain-length regulator, cld pHS-2 . LPS analysis showed that mutations in rfb genes and in a candidate rfaL gene either eliminated the entire O-antigen side chains or produced chains of greatly reduced length. Mutation in a previously unidentified gene, rfaX, affected the LPS core region and resulted in reduced amounts of O-antigen. Mutants defective in cld pHS-2 or rol had different distributions of O-antigen chain lengths. The results of tissue-culture cell invasion and plaque assays, the Seré ny test, and serum-sensitivity assay suggested roles for the different LPS synthesis genes in bacterial survival and virulence; rfaL, rfaX and rfb loci are required for serum resistance and intercellular spread, but not for invasion; cld pHS-2 is required for resistance to serum killing and for full inflammation in the Seré ny test, but not for invasion or intercellular spread, while rol is required for normal invasiveness and plaque formation, but not for serum resistance. Thus, O-antigen synthesis and chain-length regulation genes encoded on both the chromosome and the small plasmid pHS-2 play important roles in S. flexneri invasion and virulence.

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“…An a-helical configuration of LacY is supported by circular dichroic studies [108], Laser Raman spectroscopy [109] and Fourier transform infrared spectroscopy [11]; however, using the same data set, partial/3-sheet structures have also been suggested [110]. A 12-helix motif of LacY [111], MelB [112], TetA [113], UhpT [114], and several other transport proteins is supported by phoA fusion analyses [115]. For LacY and TetA, the presence of 12 transmembrane segments is further supported by limited proteolysis, immunological analyses and chemical modification studies [11,113].…”

Section: Iv-a Primary Sequence and Secondary Structurementioning

confidence: 99%