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Activation of54 -dependent gene expression essential for formation of flagella in Campylobacter jejuni requires the components of the inner membrane-localized flagellar export apparatus and the FlgSR twocomponent regulatory system. In this study, we characterized the FlgS sensor kinase and how activation of the protein is linked to the flagellar export apparatus. We found that FlgS is localized to the C. jejuni cytoplasm and that His141 of FlgS is essential for autophosphorylation, phosphorelay to the cognate FlgR response regulator, motility, and expression of 54 -dependent flagellar genes. Mutants with incomplete flagellar export apparatuses produced wild-type levels of FlgS and FlgR, but they were defective for signaling through the FlgSR system. By using genetic approaches, we found that FlgSR activity is linked to and downstream of the flagellar export apparatus in a regulatory cascade that terminates in expression of 54 -dependent flagellar genes. By analyzing defined flhB and fliI mutants of C. jejuni that form flagellar export apparatuses that are secretion incompetent, we determined that formation of the apparatus is required to contribute to the signal sensed by FlgS to terminate in activation of expression of 54 -dependent flagellar genes. Considering that the flagellar export apparatuses of Escherichia coli and Salmonella species influence 28 -dependent flagellar gene expression, our work expands the signaling activity of the apparatuses to include 54 -dependent pathways of C. jejuni and possibly other motile bacteria. This study indicates that these apparatuses have broader functions beyond flagellar protein secretion, including activation of essential two-component regulatory systems required for expression of 54 -dependent flagellar genes.Responding to changing environmental and intracellular conditions in cells requires efficient communication networks that can rapidly receive and integrate signals. Two-component regulatory systems, which are distributed almost ubiquitously among prokaryotic organisms, allow bacteria to monitor their intracellular and extracellular environments and react by altering the expression of appropriate genes. These systems are typically comprised of a sensor histidine kinase and a response regulator protein (reviewed in references 46 and 65). The sensor protein contains a domain usually in the N-terminal portion that detects a specific signal, commonly through an interaction with another protein or a small effector molecule. Activation includes autophosphorylation of the sensor kinase and a conformational change that allows the transmitter domain, usually in the C-terminal portion, to activate a cognate response regulator via phosphotransfer. Some histidine kinases also have the ability to function as a phosphatase to remove a phosphate group from either themselves or their cognate response regulators when activity of the regulatory system is not favored.The largest group of sensor histidine kinases includes those that are anchored to the cytoplasmic membrane and receiv...

Flagellar motility inFlagella are produced by diverse bacterial species to aid in processes, including motility and adhesion, that allow bacteria to occupy an environmental niche or maintain a relationship with a host. Flagellar biosynthesis requires coordinating both the expression of over 40 flagellar genes and assembly of the encoded flagellar components into the organelle. Several mechanisms of flagellar gene regulation have evolved, with the best-understood system exemplified by Escherichia coli and Salmonella species. Flagellar genes in these bacteria are grouped into three classes based on their temporal expression (reviewed in reference 10). Briefly, global regulatory signals activate the transcription of the class I (early) genes flhD and flhC, which encode the master regulator of flagellar gene transcription. FlhDC activates the transcription of class II (middle) flagellar genes, which include those encoding the hook and basal body components and the alternative sigma factor, 28 . 28 -dependent class III (late) genes include those for the flagellins and the motor complex.Species of the Vibrio and Pseudomonas genera employ a four-tiered regulatory cascade utilizing 28 and another, alternative sigma factor, 54 , to control the expression of flagellar genes (14,39,49). In Vibrio cholerae, the class I master regulator, FlrA, interacts with 54 for the transcription of class II genes, including the flrBC operon which encodes a two-component regulatory system (31). The transcription of class III genes, such as those encoding the hook, basal body, and major flagellin, is activated by FlrC and the 54 -RNA polymerase (RNAP) holoenzyme (12, 49). Class IV genes are 28 dependent and include those encoding the minor flagellin and motor proteins. Similar genetic regulators and pathways exist in Pseudomonas aeruginosa to control the transcription of flagellar genes (2,14,29,50). As in Vibrio and Pseudomonas species, the regulatory system employed by Helicobacter pylori also requires 28 and 54 , but a master regulator of flagellar gene transcription has not been described and may be absent in this bacterium (28,43).Many bacteria utilize 54 to transcribe genes required for such diverse activities as nitrogen fixation, root nodule formation during plant symbiosis, and flagellar motility (reviewed in reference 30, 35). Unlike other factors, 54 -RNAP holoenzyme alone cannot mediate the opening of DNA at target promoters. Instead, it requires interaction with a regulator (also termed "enhancer-binding protein") to mediate this process. NtrC is one such, well-characterized, 54 -dependent response regulator, consisting of a phosphorylatable N-terminal regulatory (or receiver) domain, a central 54 interaction domain, and a C-terminal domain (CTD) that contains dimerization determinants and is also indispensable for DNA binding in vivo (16; reviewed in reference 45). Under nitrogen-limiting conditions, the NtrB histidine kinase autophosphorylates and donates its phosphate residue to NtrC at residue D54 (32,44,51), which activat...

Young adult chinchillas were atraumatically inoculated with Moraxella catarrhalis via the nasal route. Detailed histopathologic examination of nasopharyngeal tissues isolated from these M. catarrhalis-infected animals revealed the presence of significant inflammation within the epithelium. Absence of similar histopathologic findings in sham-inoculated animals confirmed that M. catarrhalis was exposed to significant host-derived factors in this environment. Twenty-four hours after inoculation, viable M. catarrhalis organisms were recovered from the nasal cavity and nasopharynx of the animals in numbers sufficient for DNA microarray analysis. More than 100 M. catarrhalis genes were upregulated in vivo, including open reading frames ( M oraxella catarrhalis is a Gram-negative mucosal pathogen that has attracted increased interest within the scientific and medical communities for its role in several clinically significant human infections. The bacterium is a cause of upper respiratory tract infections including sinusitis and otitis media in healthy children (10, 17, 62). More recently, M. catarrhalis has been shown to be involved in conjunctivitis in children (9) and in acute exacerbations of chronic sinusitis in adults (11). Additionally, in adults, it is an important etiologic agent of exacerbations of chronic obstructive pulmonary disease (COPD) (54,55,62). It has been estimated that M. catarrhalis is responsible for up to 10% of exacerbations of COPD in the United States, a finding which translates into as many as 4 million infections per year (43).For M. catarrhalis to cause clinical disease, it typically must spread from its initial site of colonization in the nasopharynx into either the middle ear or the lower respiratory tract. It is believed that biofilm formation is an important event involved in colonization of the nasopharynx, and a recent study demonstrated that M. catarrhalis was present in a biofilm in the middle ear of children with chronic otitis media (25). It is likely that M. catarrhalis exists in a biofilm together with other normal flora in the nasopharynx. Until relatively recently, no studies had been performed in an in vivo environment to identify and better characterize the bacterial factors involved with colonization of the nasopharynx by M. catarrhalis. However, utilizing a chinchilla model, Luke et al. (36) demonstrated that type IV pili are important for colonization by M. catarrhalis in this animal model.Previous studies have examined the human antibody response to known surface proteins of M. catarrhalis as a surrogate for identification of bacterial genes expressed in vivo (for a representative example, see reference 42), and one study was able to detect mRNA from a small number of selected M. catarrhalis genes in nasopharyngeal secretions from young children with acute respiratory tract illness (39). The demonstration that the chinchilla nasopharynx can be colonized by M. catarrhalis (5, 36), together with the development of M. catarrhalis DNA microarrays (19,65), presented the op...

Moraxella catarrhalis is subjected to oxidative stress from both internal and environmental sources. A previous study (C. D. Pericone, K. Overweg, P. W. Hermans, and J. N. Weiser, Infect. Immun. 68:3990-3997, 2000) indicated that a wild-type strain of M. catarrhalis was very resistant to killing by exogenous hydrogen peroxide (H 2 O 2 ). The gene encoding OxyR, a LysR family transcriptional regulator, was identified and inactivated in M. catarrhalis strain O35E, resulting in an increase in sensitivity to killing by H 2 O 2 in disk diffusion assays and a concomitant aerobic serial dilution effect. Genes encoding a predicted catalase (KatA) and an alkyl hydroperoxidase (AhpCF) showed dose-dependent upregulation in wild-type cells exposed to H 2 O 2 . DNA microarray and real-time reverse transcription-PCR (RT-PCR) analyses identified M. catarrhalis genes whose expression was affected by oxidative stress in an OxyR-dependent manner. Testing of M. catarrhalis O35E katA and ahpC mutants for their abilities to scavenge exogenous H 2 O 2 showed that the KatA catalase was responsible for most of this activity in the wild-type parent strain. The introduction of the same mutations into M. catarrhalis strain ETSU-4 showed that the growth of a ETSU-4 katA mutant was markedly inhibited by the addition of 50 mM H 2 O 2 but that this mutant could still form a biofilm equivalent to that produced by its wild-type parent strain.