Multiple Peptidoglycan Modification Networks Modulate Helicobacter pylori's Cell Shape, Motility, and Colonization Potential

Helical cell shape of the gastric pathogen Helicobacter pylori has been suggested to promote virulence through viscosity-dependent enhancement of swimming velocity. However, H. pylori csd1 mutants, which are curved but lack helical twist, show normal velocity in viscous polymer solutions and the reason for their deficiency in stomach colonization has remained unclear. Characterization of new rod shaped mutants identified Csd4, a DL-carboxypeptidase of peptidoglycan (PG) tripeptide monomers and Csd5, a putative scaffolding protein. Morphological and biochemical studies indicated Csd4 tripeptide cleavage and Csd1 crosslinking relaxation modify the PG sacculus through independent networks that coordinately generate helical shape. csd4 mutants show attenuation of stomach colonization, but no change in proinflammatory cytokine induction, despite four-fold higher levels of Nod1-agonist tripeptides in the PG sacculus. Motility analysis of similarly shaped mutants bearing distinct alterations in PG modifications revealed deficits associated with shape, but only in gel-like media and not viscous solutions. As gastric mucus displays viscoelastic gel-like properties, our results suggest enhanced penetration of the mucus barrier underlies the fitness advantage conferred by H. pylori's characteristic shape

Characterization of the sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 as a member of the metallo(zinc) carboxypeptidase A family. Modular design of the protein.

The sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 has been analysed by proton-induced X-ray emission. It contains 1 equivalent Zn2+ per mol of protein. As derived from gene cloning and sequencing, the B. sphaericus Zn peptidase I is a two-module protein. A 100-amino-acid-residue N-terminal domain consisting of two tandem segments of similar sequences, is fused to a 296-amino-acid-residue C-terminal catalytic domain. The catalytic domain belongs to the Zn carboxypeptidase A family, the closest match being observed with the Streptomyces griseus carboxypeptidase [Narahashi (1990) J. Biochem. 107, 879-886] and with the family prototype, bovine carboxypeptidase A. The catalytic domain of the B. sphaericus peptidase I possesses, distributed along the amino-acid sequence, peptide segments, a triad His162-Glu165-His307 and a dyad Tyr347-Glu366 that are equivalent to secondary structures, the zinc-binding triad His69-Glu72-His196 and the catalytic dyad Tyr248-Glu270 of bovine carboxypeptidase A respectively. The N-terminal repeats of the B. sphaericus peptidase I have similarity with the C-terminal repeats of the Enterococcus hirae muramidase 2, the Streptococcus (now Enterococcus) faecalis autolysin and the Bacillus phi PZA and phi 29 lysozymes, to which a role in the recognition of a particular moiety of the bacterial cell envelope has been tentatively assigned. Detergents enhance considerably the specific activity of the B. sphaericus peptidase I

Cloning, Sequencing, and Characterization of the Iturin A Operon

Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are d-Ser→l-Asn, while in iturin A these amino acids are inverted (i.e., d-Asn→l-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold