In 1995, the Institute for Genomic Research completed the genome sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. Although extremely useful in understanding the basic biology of H. influenzae, these data have not provided significant insight into disease caused by nontypeable H. influenzae, as serotype d strains are not pathogens. In contrast, strains of nontypeable H. influenzae are the primary pathogens of chronic and recurrent otitis media in children. In addition, these organisms have an important role in acute otitis media in children as well as other respiratory diseases. Such strains must therefore contain a gene repertoire that differs from that of strain Rd. Elucidation of the differences between these genomes will thus provide insight into the pathogenic mechanisms of nontypeable H. influenzae. The genome of a representative nontypeable H. influenzae strain, 86-028NP, isolated from a patient with chronic otitis media was therefore sequenced and annotated. Despite large regions of synteny with the strain Rd genome, there are large rearrangements in strain 86-028NP's genome architecture relative to the strain Rd genome. A genomic island similar to an island originally identified in H. influenzae type b is present in the strain 86-028NP genome, while the mu-like phage present in the strain Rd genome is absent from the strain 86-028NP genome. Two hundred eighty open reading frames were identified in the strain 86-028NP genome that were absent from the strain Rd genome. These data provide new insight that complements and extends the ongoing analysis of nontypeable H. influenzae virulence determinants.In 1995 Haemophilus influenzae strain Rd, a rough derivative of H. influenzae serotype d strain KW20 (strain Rd hereafter), became the first free-living organism to have its genome sequenced to completion (34). Importantly, this also helped establish the large-scale shotgun approach, mated with the utilization of a scaffolding library and computer-assisted assembly, as a rational and expeditious approach for the sequencing of small bacterial genomes. Strain Rd was chosen as the prototypic bacterium for complete genome sequencing as it has a genome size representative of other bacteria and a GϩC content close to that of the human genome. Additionally, at the time of sequencing, a physical map of the strain Rd genome did not exist, so this genome was a good test for the approach of shotgun sequencing, scaffolding, and assembly (34).Although strain Rd is the exemplar organism for the current small-genome sequencing rationale and an important model organism for studying H. influenzae biology, strain Rd is a poor model for the study of pathogenicity caused by members of the genus Haemophilus. Serotype b strains of H. influenzae cause invasive diseases, for example, meningitis, and nontypeable H. influenzae (NTHi) strains principally have a role in localized respiratory disease, particularly in otitis media, acute sinusitis, and community-acquired pneumonia and have important conseque...

The cytoplasmic dynein light chain Tctex1 is a candidate for one of the distorter products involved in the non-Mendelian transmission of mouse t haplotypes. It has been unclear, however, how the t-specific mutations in this protein, which is found associated with cytoplasmic dynein in many tissues, could result in a male germ cell–specific phenotype. Here, we demonstrate that Tctex1 is not only a cytoplasmic dynein component, but is also present both in mouse sperm and Chlamydomonas flagella. Genetic and biochemical dissection of the Chlamydomonas flagellum reveal that Tctex1 is a previously undescribed component of inner dynein arm I1. Combined with the recent identification of another putative t complex distorter, Tctex2, within the outer dynein arm, these results support the hypothesis that transmission ratio distortion (meiotic drive) of mouse t haplotypes involves dysfunction of both flagellar inner and outer dynein arms but does not require the cytoplasmic isozyme.

Haemophilus influenzae is considered a nonmotile organism that expresses neither flagella nor type IV pili, although H. influenzae strain Rd possesses a cryptic pilus locus. We demonstrate here that the homologous gene cluster pilABCD in an otitis media isolate of nontypeable H. influenzae strain 86-028NP encodes a surface appendage that is highly similar, structurally and functionally, to the well-characterized subgroup of bacterial pili known as type IV pili. This gene cluster includes a gene (pilA) that likely encodes the major subunit of the heretofore uncharacterized H. influenzae-expressed type IV pilus, a gene with homology to a type IV prepilin peptidase (pilD) as well as two additional uncharacterized genes (pilB and pilC). A second gene cluster (comABCDEF) was also identified by homology to other pil or type II secretion system genes. When grown in chemically defined medium at an alkaline pH, strain 86-028NP produces approximately 7-nm-diameter structures that are near polar in location. Importantly, these organisms exhibit twitching motility. A mutation in the pilA gene abolishes both expression of the pilus structure and the twitching phenotype, whereas a mutant lacking ComE, a Pseudomonas PilQ homologue, produced large appendages that appeared to be membrane bound and terminated in a slightly bulbous tip. These latter structures often showed a regular pattern of areas of constriction and expansion. The recognition that H. influenzae possesses a mechanism for twitching motility will likely profoundly influence our understanding of H. influenzae-induced diseases of the respiratory tract and their sequelae.

The M r 8,000 light chain originally identified in Chlamydomonas flagellar dynein is also a component of both cytoplasmic dynein and myosin V. Furthermore, this small protein has been implicated as an inhibitor of neuronal nitric oxide synthase, suggesting that it may play multiple regulatory roles within the cell. Covalent cross-linking of both dynein and myosin V using 1,5-difluoro-2,4-dinitrobenzene revealed that this light chain exists as a dimer in situ. This observation was confirmed using two additional amine-selective crosslinking reagents (dimethyl pimelimidate and disuccinimidyl suberate). When expressed as a C-terminal fusion with maltose-binding protein, the presence of the light chain caused the recombinant molecule to dimerize. Analysis of fusions containing truncated light chains identified the predicted amphiphilic helix (residues 14 -32) as sufficient to cause dimerization; cross-linking required a second helical segment (residues 33-46). Together the data presented suggest that two light chains interact to form a parallel dimeric structure. This arrangement has significant implications for the potential functions of this highly conserved molecule and suggests a mechanism by which it might dissociate nitric oxide synthase.Dyneins are highly complex molecular motors that translocate the cargo to which they are attached along microtubules (see Refs. 1 and 2 for review). Molecular analysis of components from both cytoplasmic and flagellar outer arm dyneins has revealed that these microtubule motors share a M r 8,000 polypeptide (actual mass ϭ 10.3 kDa) that has been very highly conserved throughout evolution (ϳ90% sequence identity between the Chlamydomonas and human proteins) (3, 4). In Drosophila, partial loss-of-function mutations in this protein lead to morphogenetic defects in bristle and wing development, female sterility, and disruption of sensory axon trajectories; total loss induces apoptosis and causes embryonic lethality (5, 6). Mutation of the homologous protein in Aspergillus results in a nuclear migration phenotype similar to that observed with dynein heavy chain null mutants (7).Intriguingly, fractionation of whole mammalian brain extracts revealed at least three biochemically distinct pools of this protein: (i) non-microtubule-associated; (ii) microtubuleassociated, eluted with ATP or salt; and (iii) microtubule-associated, not salt-extractable (4). Only the second pool was cytoplasmic dynein-associated, and indeed, the predominant form of this molecule (pool (i)) did not cosediment with microtubules. Likewise, this polypeptide is associated with at least two distinct components of the Chlamydomonas flagellar axoneme. There are 8 -10 copies of this protein within the outer dynein arm (8); this fraction is absent in axonemes derived from mutants that lack this structure (9), and it may be extracted from wild-type axonemes with 0.6 M NaCl. The second flagellar fraction, which accounts for ϳ50% of the total axonemal M r 8,000 LC, 1 is tightly integrated into the axonemal superstructure...