An Empirical Strategy for Characterizing Bacterial Proteomes across Species in the Absence of Genomic Sequences

Abstract

Global protein identification through current proteomics methods typically depends on the availability of sequenced genomes. In spite of increasingly high throughput sequencing technologies, this information is not available for every microorganism and rarely available for entire microbial communities. Nevertheless, the protein-level homology that exists between related bacteria makes it possible to extract biological information from the proteome of an organism or microbial community by using the genomic sequences of a near neighbor organism. Here, we demonstrate a trans-organism search strategy for determining the extent to which near-neighbor genome sequences can be applied to identify proteins in unsequenced environmental isolates. In proof of concept testing, we found that within a CLUSTAL W distance of 0.089, near-neighbor genomes successfully identified a high percentage of proteins within an organism. Application of this strategy to characterize environmental bacterial isolates lacking sequenced genomes, but having 16S rDNA sequence similarity to Shewanella resulted in the identification of 300–500 proteins in each strain. The majority of identified pathways mapped to core processes, as well as to processes unique to the Shewanellae, in particular to the presence of c-type cytochromes. Examples of core functional categories include energy metabolism, protein and nucleotide synthesis and cofactor biosynthesis, allowing classification of bacteria by observation of conserved processes. Additionally, within these core functionalities, we observed proteins involved in the alternative lactate utilization pathway, recently described in Shewanella.

Extracellular polymeric substances from Shewanella sp. HRCR‐1 biofilms: characterization by infrared spectroscopy and proteomics

“…As the genome of Shewanella sp. HRCR‐1 has not been sequenced, peptides were identified by searching against in silico tryptic sequences derived from in‐house S. oneidensis MR‐1, MR‐4 and MR‐7 protein FASTA protein sequences (Turse et al ., 2010). The results showed that a total of 425 ( S. oneidensis MR‐1), 323 ( S. oneidensis MR‐4) and 316 ( S. oneidensis MR‐7) proteins were identified with high confidence (at least two unique peptides) in the EPS samples (Table S2).…”

Characterization of microbial mixtures by mass spectrometry

“…One bacterial isolate has been assigned to genus Ruegeria after a rapid MALDI MS screening of 30 seawater bacterial isolates, demonstrating the usefulness of this combined proteogenomic approach for environmental monitoring of complex environments. A trans‐organism composite proteomics strategy has been applied to characterize unsequenced environmental bacterial isolates similar to Shewanella , as inferred from 16S ribosome comparison . Up to 500 Shewanella proteins, mapped to core pathway processes, have been identified in each unsequenced strain.…”

Microbial metaproteomics: identifying the repertoire of proteins that microorganisms use to compete and cooperate in complex environmental communities

“…While the field is too vast to adequately detail in this limited review, it is worth noting that some of the basic database searching and data extraction methodologies have become quite standardized and fairly routine, leaving more attention to be paid to data mining and analyses. This has led to a noticeable increase in the number of interdisciplinary interactions between experimental and computational researchers, often focused on design of an integrated approach for specific problems, such as characterizing proteomes across microbial species in the absence of specific genomic data [28], or evaluating new approaches to integrate metagenomic and metaproteomics information [29,30].…”