The emb genes are conserved among different mycobacteria. In Mycobacterium smegmatis and Mycobacterium tuberculosis, they belong to an operon comprising three genes, embC, embA, and embB. The EmbB protein has been proposed to be the target of ethambutol, a drug which is known to inhibit the synthesis of the arabinan portion of the mycobacterial cell wall arabinogalactan (AG). To further define the role of EmbB protein in arabinan biosynthesis, embA, -B, and -C genes were inactivated individually by homologous recombination in M. smegmatis. All three mutants were viable, and among the three, the slowest growing embB ؊ mutant encountered profound morphological changes and exhibited a higher sensitivity to hydrophobic drugs and detergents, presumably due to an increase in cell wall permeability. Furthermore, chemical analyses showed that there was a diminution in the arabinose content of arabinogalactan from the embA ؊ and embB ؊ mutants. Specifically, in comparison with the wild-type strain, the crucial terminal hexaarabinofuranosyl motif, which is a template for mycolylation, was altered in both embA ؊ and embB

Francisella tularensis is a highly infectious bacterial pathogen, responsible for the zoonotic disease tularemia. We screened a bank of transposon insertion mutants of F. tularensis subsp. holarctica LVS for colony morphology alterations and selected a mutant with a transposon insertion in wbtA, the first gene of the predicted lipopolysaccharide O-antigen gene cluster. Inactivation of wbtA led to the complete loss of O antigen, conferred serum sensitivity, impaired intracellular replication, and severely attenuated virulence in the mouse model. Notably, this mutant afforded protection against a challenge against virulent LVS.

Fimbriae have been shown to play an essential role in the adhesion of pathogenic gram-negative bacteria to host cells. In the enteroinvasive bacterium Yersinia pseudotuberculosis, we characterized a previously unknown 11-kb chromosomal locus involved in the synthesis of type IV pili. The locus consists of 11 open reading frames forming a polycistronic unit and encoding putative Pil proteins, PilLMNOPQRSUVW. When introduced into Escherichia coli, the Y. pseudotuberculosis operon reconstituted bundles of filaments at a pole on the bacterial surface, demonstrating that the pil locus was functional in a heterogenous genetic background. Environmental factors regulated transcription of the Y. pseudotuberculosis operon; in particular, temperature, osmolarity, and oxygen tension were critical cues. Deletion of the type IV pilus gene cluster was associated with a reduction of Y. pseudotuberculosis pathogenicity for mice infected orally. Forty-one percent of Y. pseudotuberculosis strains isolated from human or animal sources harbored the type IV pilus locus. Therefore, the pil locus of Y. pseudotuberculosis might constitute an "adaptation island," permitting the microorganism to colonize a vast reservoir.Type IV pili are appendages emanating from the surfaces of several gram-negative bacteria, including species pathogenic for animals and plants (Actinobacillus pleuropneumoniae, Aeromonas veronii biovar sobria, Aeromonas caviae, Dichelobacter nodosus, Eikenella corrodens, enteropathogenic Escherichia coli [EPEC] and enterotoxigenic E. coli [ETEC], Legionella pneumophila, Moraxella bovis, Neisseria meningitidis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella enterica, and Vibrio cholerae) (9,11,12,15,16,18,19,20,21,25,27,40,45,49). These structures, which may be peritrichous or polar on the cell surface, sometimes form bundles and have been implicated in a variety of bacterial functions, including cell adhesion (24), bacteriophage adsorption (17, 19), plasmid transfer (19), and twitching motility, a form of flagellum-independent locomotion (26, 44). Pili are composed of pilin subunits, the primary structures of which are conserved (24). All pilins are produced from prepilin molecules through cleavage of the leader sequence by a prepilin peptidase. There is a long hydrophobic segment (20 to 30 amino acid residues [aa]) at the N-terminal region of the mature pilin protein. Type IV pili are classified into two subclasses, IVA and IVB, on the basis of similarities in the deduced amino acid sequences of prepilins. Type IVA prepilins have a very short leader sequence (5 to 6 aa), and the N-terminal amino acid of the mature pilin is a methylated phenylalanine. Type IVB prepilins tend to have longer signal sequences (13 to 30 aa), and the N-terminal amino acid of the mature protein may be a methionine (methylated in some cases), leucine, tryptophan, or serine. The assembly machinery involved in the formation of fimbriae consists of a set of proteins encoded by genes either scattered throughout the bacterial genome (IVA ...