Identification of a bacteriocin and its cognate immunity factor expressed by Moraxella catarrhalis

<p>Abstract</p> <p>Background</p> <p>Bacteriocins are antimicrobial proteins and peptides ribosomally synthesized by some bacteria which can effect both intraspecies and interspecies killing.</p> <p>Results</p> <p><it>Moraxella catarrhalis </it>strain E22 containing plasmid pLQ510 was shown to inhibit the growth of <it>M. catarrhalis </it>strain O35E. Two genes (<it>mcbA </it>and <it>mcbB</it>) in pLQ510 encoded proteins predicted to be involved in the secretion of a bacteriocin. Immediately downstream from these two genes, a very short ORF (<it>mcbC</it>) encoded a protein which had some homology to double-glycine bacteriocins produced by other bacteria. A second very short ORF (<it>mcbI</it>) immediately downstream from <it>mcbC </it>encoded a protein which had no significant similarity to other proteins in the databases. Cloning and expression of the <it>mcbI </it>gene in <it>M. catarrhalis </it>O35E indicated that this gene encoded the cognate immunity factor. Reverse transcriptase-PCR was used to show that the <it>mcbA</it>, <it>mcbB</it>, <it>mcbC</it>, and <it>mcbI </it>ORFs were transcriptionally linked. This four-gene cluster was subsequently shown to be present in the chromosome of several <it>M. catarrhalis </it>strains including O12E. Inactivation of the <it>mcbA</it>, <it>mcbB</it>, or <it>mcbC </it>ORFs in <it>M. catarrhalis </it>O12E eliminated the ability of this strain to inhibit the growth of <it>M. catarrhalis </it>O35E. In co-culture experiments involving a <it>M. catarrhalis </it>strain containing the <it>mcbABCI </it>locus and one which lacked this locus, the former strain became the predominant member of the culture after overnight growth in broth.</p> <p>Conclusion</p> <p>This is the first description of a bacteriocin and its cognate immunity factor produced by <it>M. catarrhalis</it>. The killing activity of the McbC protein raises the possibility that it might serve to lyse other <it>M. catarrhalis </it>strains that lack the <it>mcbABCI </it>locus, thereby making their DNA available for lateral gene transfer.</p

Identification of Gene Products Involved in the Oxidative Stress Response of Moraxella catarrhalis ▿ †

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 (H2O2). 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 H2O2 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 H2O2. 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 H2O2 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 H2O2 but that this mutant could still form a biofilm equivalent to that produced by its wild-type parent strain

Moraxella catarrhalis Synthesizes an Autotransporter That Is an Acid Phosphatase▿

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10−10) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity