Metabolic analysis of Moraxella catarrhalis and the effect of selected in vitro growth conditions on global gene expression

Abstract

The nucleotide sequence from the genome of Moraxella catarrhalis ATCC 43617 was annotated and used both to assess the metabolic capabilities and limitations of this bacterium and to design probes for a DNA microarray. An absence of gene products for utilization of exogenous carbohydrates was noteworthy and could be correlated with published phenotypic data. Gene products necessary for aerobic energy generation were present, as were a few gene products generally ascribed to anaerobic systems. Enzymes for synthesis of all amino acids except proline and arginine were present. M. catarrhalis DNA microarrays containing 70-mer oligonucleotide probes were designed from the genome-derived nucleotide sequence data. Analysis of total RNA extracted from M. catarrhalis ATCC 43617 cells grown under iron-replete and iron-restricted conditions was used to establish the utility of these DNA microarrays. These DNA microarrays were then used to analyze total RNA from M. catarrhalis cells grown in a continuous-flow biofilm system and in the planktonic state. The genes whose expression was most dramatically increased by growth in the biofilm state included those encoding a nitrate reductase, a nitrite reductase, and a nitric oxide reductase. Real-time reverse transcriptase PCR analysis was used to validate these DNA microarray results. These results indicate that growth of M. catarrhalis in a biofilm results in increased expression of gene products which can function not only in energy generation but also in resisting certain elements of the innate immune response.

FIG. 1.

Effect of iron limitation on growth and protein expression by M. catarrhalis ATCC 43617. (A) Growth of strain ATCC 43617 in BHI broth containing various amounts (10 to 50 μM) of Desferal. (B) Western blot analysis of CopB protein expression by this strain grown in BHI medium in the presence of no Desferal (lane 1), 10 μM Desferal (lane 2), 30 μM Desferal (lane 3), and 50 μM Desferal (lane 4). Proteins in whole-cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and probed with the CopB-specific monoclonal antibody 10F3 (2). The positions of molecular weight markers are indicated on the left.

FIG. 2.

Use of real-time RT-PCR to verify DNA microarray results. Expression of 20 selected genes in M. catarrhalis ATCC 43617 cells grown in a biofilm and in the planktonic state was measured by real-time RT-PCR analysis as described in Materials and Methods. The logarithm of the difference in gene expression between biofilm and planktonic cells as determined by real-time RT-PCR is plotted adjacent to the results obtained in the DNA microarray analysis.