Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.

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The two major molecular chaperone families that mediate ATP-dependent protein folding and refolding are the heat shock proteins Hsp6Os (GroEL) and Hsp7Os (DnaK). Cip proteins, like chaperones, are highly conserved, present in all organisms, and contain ATP and polypeptide binding sites. We discovered that CipA, the ATPase component of the ATP-dependent ClpAP protease, is a molecular chaperone. CipA performs the ATP-dependent chaperone function of DnaK and DnaJ in the in vitro activation of the plasmid P1 RepA replication initiator protein. RepA is activated by the conversion of dimers to monomers. We show that CipA targets RepA for degradation by ClpP, demonstrating a direct link between the protein unfolding function of chaperones and proteolysis. In another chaperone assay, ClpA protects luciferase from irreversible heat inactivation but is unable to reactivate luciferase.Molecular chaperones interact with other proteins to mediate ATP-dependent protein folding, refolding, assembly, and disassembly of proteins. The Hsp7O chaperone system of Escherichia coli consists of the DnaK, DnaJ, and GrpE heat shock proteins. In vivo these three heat shock proteins function together in many cellular processes, as demonstrated by the observations that mutants in dnaK, dnaJ, and grpE have similar effects on DNA replication of E. coli, plasmids P1 and F and phage A, RNA synthesis, cell division, protein transport, regulation of the heat shock response, protection of enzymes from misfolding or aggregation during heat shock, and degradation of abnormal proteins (reviewed in ref.

DnaK is a major heat shock protein of Escherichia coli and the homolog of hsp7O in eukaryotes. We demonstrate the mechanism by which DnaK and another heat shock protein, DnaJ, render the plasmid P1 initiator RepA 100-fold more active for binding to the P1 origin of replication.

Heat-shock proteins are normal constituents of cells whose synthesis is increased on exposure to various forms of stress. They are interesting because of their ubiquity and high conservation during evolution. Two families of heat-shock proteins, hsp60s and hsp70s, have been implicated in accelerating protein folding and oligomerization and also in maintaining proteins in an unfolded state, thus facilitating membrane transport. The Escherichia coli hsp70 analogue, DnaK, and two other heat-shock proteins, DnaJ and GrpE, are required for cell viability at high temperatures and are involved in DNA replication of phage lambda and plasmids P1 and F. These three proteins are involved in replication in vitro of P1 DNA along with many host replication proteins and the P1 RepA initiator protein. RepA exists in a stable protein complex with DnaJ containing a dimer each of RepA and DnaJ. We report here that DnaK and DnaJ mediate an alteration in the P1 initiator protein, rendering it much more active for oriP1 DNA binding.

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The thermodynamics of the binding of cyclic adenosine monophosphate (cAMP) and its non-functional analog, cyclic guanosine monophosphate (cGMP), to cyclic AMP receptor protein (CRP) and its T127L mutant were investigated by isothermal titration calorimetry (

Roy etal. [Roy, A., Haziza, C. & Danchin, A. (1983) EMBO J. 2,[791][792][793][794][795][796][797] established that translation of Escherichia coli adenylate cyclase initiates at a UUG codon, and they suggested this might decrease the efficiency of translation. We investigated the effect of varying the initiation codon on the expression of the adenylate cyclase (cya) gene. Using oligonucleotide-directed mutagenesis, we changed the UUG initiation codon to GUG and the more common initiator AUG and assayed for cya gene expression in a number of ways. First, the GUG initiation codon, in place of UUG, doubled cya expression when cya was expressed from the dual cya P1/P2 promoters. The corresponding AUG codon construct was nonviable. Second, when the cya gene was placed under the transcriptional control of the thermoinducible phage A PL promoter, the relative amounts of cya gene product were 1:2:6 for the UUG, GUG, and AUG initiation codons, respectively. Finally, the cya P2 promoter, Shine-Dalgarno sequence, and the DNA corresponding to the first 86 codons of cya were fused to DNA encoding the E. coli galactokinase gene beginning at the second codon. The relative amounts of the fusion polypeptides, which had galactokinase activity, were 1:2:3 for the UUG, GUG, and AUG initiation codons, respectively. These results demonstrate that the cya UUG initiation codon limits cya expression at the level of translation.Translation initiation requires an initiation codon and a properly spaced sequence upstream of the initiation codon that has base pairing potential with the 3' end of the 16S ribosomal RNA (the Shine and Dalgarno or SD sequence) (1, 2). AUG, GUG, and UUG triplets have been shown to stabilize the binding of initiator fMet-tRNA to ribosomes in vitro, suggesting that these three codons may function in vivo as initiation codons (3). Comparison of protein sequence data with the corresponding nucleic acid sequence data for more than 100 genes has shown that, in vivo, all eukaryotic and nearly all prokaryotic genes use the translation initiation codon AUG. However, there are 9 examples of GUG and 3 examples of UUG functioning as initiation codons in Escherichia coli (2, 4). The gene for E. coli adenylate cyclase (cya), the enzyme that synthesizes the important cellular regulator cAMP, is one of the genes that uses the unusual UUG initiation codon (4-6).We have investigated the effect of this unusual initiation codon on cya expression by changing the DNA sequence coding for the UUG initiation codon to ATG and GTG, using oligonucleotide-directed mutagenesis. A comparison of the activities associated with the three codons was made in three different environments: (i) in the normal cya environment, with the cya gene expressed from the dual cya P1/P2 promoters, (it) in a transcription fusion with the cya gene under the transcriptional control of the phage X PL promoter, and (iii) in a gene fusion with the cya gene fused to the E. coli galactokinase (galK) gene to generate a fusion protein with galactokinase activity....