The deduced metabolism of M. catarrhalis was used to assess the completeness of the available nucleotide sequence from the genome of M. catarrhalis ATCC 43617 and to provide a context for interpreting transcriptome profiles. In addition, a comparison of the metabolism of M. catarrhalis with that of two other gram-negative colonizers of the nasopharynx, H. influenzae and N. meningitidis, has the potential to provide some insight into metabolic approaches used by these three organisms to survive in the environment of the nasopharynx. Table presents some key metabolic features of these three pathogens, with an emphasis on significant differences.
Comparison of some of the metabolic properties of M. catarrhalis, H. influenzae, and N. meningitidisa
At the outset, it is readily apparent that these organisms utilize different metabolic strategies for survival in the nasopharynx. All three organisms grow readily under aerobic conditions, while S. pneumoniae
is an aerotolerant anaerobe. H. influenzae
can grow anaerobically in the apparent absence of alternative respiratory substrates (e.g., nitrate) (20
), and both growth experiments (21
) and analysis of encoded protein products of the H. influenzae
Rd genome (69
) indicated that this organism might prefer growth under reducing conditions. N. meningitidis
cannot grow under strictly anaerobic conditions but has effective systems for using both nitrite and nitric oxide as respiratory substrates (5
). Similar to the meningococcus, M. catarrhalis
cannot grow anaerobically (4
). However, in addition to containing the genes encoding predicted nitrite and nitric oxide reductases (Table ), M. catarrhalis
also possesses the ability to reduce nitrate to nitrite (16
). Interestingly, during growth in a biofilm, these genes encoding the enzymatic machinery necessary to reduce nitrate to the level of nitrous oxide were among those that were most highly expressed (Table ).
differs substantially from the other two pathogens with respect to central metabolic pathways. M. catarrhalis
is unable to utilize exogenous carbohydrates (16
), apparently lacking both glycolytic pathways and sugar transport systems (Table ). In contrast, both H. influenzae
and N. meningitidis
can utilize a limited number of carbohydrates (16
), which is consistent with the presence of intact glycolytic pathways and complete phosphotransferase sugar transport systems in these two organisms. In aggregate, these organisms can utilize only a limited variety of carbohydrates, which suggests that the nasopharynx may be restricted in carbohydrate diversity and perhaps availability. Only N. meningitidis
appears to have a complete citric acid cycle. M. catarrhalis
appears to lack both subunits of succinyl coenzyme A synthetase, whereas H. influenzae
is missing several genes encoding citric acid cycle enzymes. Finally, M. catarrhalis
has a glyoxylate cycle, whereas the other two organisms do not (Table ).
Each of these organisms has certain nutritional deficiencies, which is not surprising considering their relatively small genomes. All three bacteria are missing crucial components for high-affinity ammonia assimilation but appear to possess low-affinity ammonia assimilation capability via the activity of glutamate dehydrogenase. However, M. catarrhalis
has been reported to be unable to assimilate ammonia (38
), which implies that M. catarrhalis
is effectively a glutamate auxotroph. The absence of high-affinity ammonia assimilation in N. meningitidis
and H. influenzae
also implies that there is glutamate auxotrophy in the nasopharynx, unless a high concentration of ammonia is present. Given that M. catarrhalis
requires at least arginine for growth and is unable to utilize carbohydrates, it seems likely that amino acids are available in the nasopharynx for the amino acid auxotrophies and energy, although the source of amino acids in the nasopharynx is not readily apparent. This conclusion is reinforced by the similar properties of N. meningitidis
and H. influenzae
: a limited ability to utilize carbohydrates, at least one requirement for an amino acid, and a potential glutamate auxotrophy in the absence of sufficient ammonia.
When total RNA extracted from M. catarrhalis
cells grown under both iron-replete and iron-limiting conditions was subjected to DNA microarray analysis using probes derived from M. catarrhalis
ATCC 43617, the genes that were markedly up-regulated included those previously shown by protein expression measurements (3
) to be affected by the availability of iron in the growth environment. The DNA microarrays were then used to identify M. catarrhalis
genes whose expression was affected by growth in a biofilm. To date, there are only very limited data available about biofilm development by M. catarrhalis
). Similarly, there is limited information about biofilm formation by N. meningitidis
), although genes induced or up-regulated by contact of this pathogen with human cells in an in vitro system have been identified by DNA microarray analysis (28
). Studies of biofilm formation by H. influenzae
are more extensive, and several gene products of this pathogen which are involved in or affected by biofilm growth have been identified (27
). One of the H. influenzae
gene products that is up-regulated by growth in a biofilm is the thiol-dependent peroxidase peroxiredoxin-glutaredoxin, and isogenic H. influenzae
mutants unable to express this protein were shown to be deficient in biofilm formation in vitro (52
). A similar peroxiredoxin was also up-regulated during growth of M. catarrhalis
in a biofilm (MCORF783) (Table ), although mutant analysis of this gene product was not performed in the present study.
In a preliminary effort to extend our findings with the DNA microarray-derived data and determine whether genes maximally up-regulated during biofilm growth were essential for this mode of growth, we inactivated two of the genes (narG and narH) which were among those most highly up-regulated by growth in a biofilm (Table ). However, when tested in a competitive index experiment in the Sorbarod continuous-flow biofilm system, the narGH mutant did not appear to have a deficiency in the ability to form biofilms (data not shown). This result suggests that nitrate reductase activity, while substantially up-regulated during biofilm growth, is not essential for biofilm development in this model system as used in this study. The up-regulation of these particular genes may instead reflect some type of sensing of reduced oxygen tension in the biofilm state.
We also noted that expression of the ORFs predicted to encode nitrite reductase and nitric oxide reductase was highly up-regulated in the biofilm (Table ). The ability of N. meningitidis
to survive in nasopharyngeal tissue has been shown to be enhanced by nitric oxide detoxification systems (67
), and in Pseudomonas aeruginosa
nitric oxide is involved in signaling biofilm dispersal (8
). The predicted ability of M. catarrhalis
to reduce nitrate to the level of nitrous oxide may provide an alternative means for energy generation by this organism under oxygen-limited conditions. In addition, the ability to reduce nitric oxide may also provide M. catarrhalis
with some level of protection against macrophage-generated nitric oxide. In this context, it is interesting to note that there has been one report describing the selection of M. catarrhalis
variants or mutants that were more resistant to nitric oxide than their wild-type parent strain (45
), but the identity of the relevant gene product(s) was not determined.
In summary, this study used nucleotide sequence data from the genome of M. catarrhalis ATCC 43617 to provide a preliminary analysis of M. catarrhalis metabolism and to construct DNA microarrays that were used to evaluate global gene expression under defined conditions in vitro. The resultant data indicate that growth of M. catarrhalis ATCC 43617 in a biofilm in vitro differentially affected the expression of genes in only a relatively few categories. The genes whose expression was most highly up-regulated were associated with energy generation involving the reduction of nitrate, nitrite, and nitric oxide. Expression of ribosomal genes was down-regulated, which is consistent with slower growth, while some heat shock genes had increased expression, which would be consistent with stress. More extensive genetic analyses are required to determine which M. catarrhalis genes are specifically required for biofilm formation in the continuous-flow system.