capsulatus Bath and ammonia-oxidizing bacteria (Klotz et al, 200

capsulatus Bath and ammonia-oxidizing bacteria (Klotz et al., 2008; Poret-Peterson et al., 2008). Deduced partial protein

Rapamycin sequences of HaoA (GenBank accession: ACV74398 and ACV74400) and HaoB (GenBank accession: ACV74399 and ACV74401) from the two M. album strains differed only in one (A55E) and two (Q95R, P111S) amino acid residues, respectively (the first amino acid residue and number reflect its position in protein sequences deduced from the pertinent genes in the genome sequence of M. album strain BG8; AFJF00000000). A blastp search revealed that sequences of the closest homologues for both proteins in Methylomonas sp. strain 16a were quite different from those in M. album strains (Table 1). As previously recognized in analysis of sequences from ammonia-oxidizing bacteria (Klotz et al., 2008), the predicted M. album HaoA sequences from methanotrophic bacteria were more identical/similar to one another than were their HaoB protein sequences (Table 1). Analysis of the deduced HaoA protein sequences allowed

detection of all necessary structural features for assembly into a functional trimeric HAO complex (Igarashi et al., 1997; Klotz et al., 2008). The haoB gene is cotranscribed with haoA in M. capsulatus Bath (Poret-Peterson et al., 2008) and Nitrosococcus oceani (Graham et Alectinib nmr al., 2011). blast searches (February 15, 2011) with haoB genes from M. capsulatus Bath or N. oceani as queries retrieved sequences only from bacterial genomes that also encoded haoA genes adjacently upstream. All haoB genes yet examined contain a palindromic sequence at the 5′-region capable of forming a leaky terminator during transcription. This is supported by the drop-off in steady-state transcript levels when comparing transcripts in M. capsulatus Bath detected

with a primer pair that targets haoAB upstream vs. haoB downstream of the palindrome (Poret-Peterson et al. 2008). Similar results were also obtained studying haoAB gene expression in N. oceani strain ATCC 19707 (M.A. Campbell & M.G. Klotz, unpublished data’). Interestingly, this palindromic sequence is part of the haoB gene segment BCKDHB encoding the N-terminal transmembrane-spanning domain immediately succeeding the signal peptide (http://www.cbs.dtu.dk/services/TMHMM/). While a function of the putative HaoB protein is still elusive, its proposed location as a periplasmic, membrane-associated protein (http://psort.hgc.jp/form.html) likely provided the functional pressure needed to conserve its N-terminal sequence and thus the palindrome. All identified haoB homologues, including those of the two M. album strains, share low conservation with only three regions of <30 bp at >60% nucleic acid sequence identity over the entire gene.

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