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Deep-sea Bathymodiolus mussels and their chemoautotrophic symbionts are well-studied representatives of mutualistic host–microbe associations. However, how host–symbiont interactions vary on the molecular level between related host and symbiont species remains unclear. Therefore, we compared the host and symbiont metaproteomes of Pacific B. thermophilus, hosting a thiotrophic symbiont, and Atlantic B. azoricus, containing two symbionts, a thiotroph and a methanotroph. We identified common strategies of metabolic support between hosts and symbionts, such as the oxidation of sulfide by the host, which provides a thiosulfate reservoir for the thiotrophic symbionts, and a cycling mechanism that could supply the host with symbiont-derived amino acids. However, expression levels of these processes differed substantially between both symbioses. Backed up by genomic comparisons, our results furthermore revealed an exceptionally large repertoire of attachment-related proteins in the B. thermophilus symbiont. These findings imply that host–microbe interactions can be quite variable, even between closely related systems.

The hydrothermal vent tube worm Riftia pachyptila lives in intimate symbiosis with intracellular sulfur-oxidizing gammaproteobacteria. Although the symbiont population consists of a single 16S rRNA phylotype, bacteria in the same host animal exhibit a remarkable degree of metabolic diversity: They simultaneously utilize two carbon fixation pathways and various energy sources and electron acceptors. Whether these multiple metabolic routes are employed in the same symbiont cells, or rather in distinct symbiont subpopulations, was unclear. As Riftia symbionts vary considerably in cell size and shape, we enriched individual symbiont cell sizes by density gradient centrifugation in order to test whether symbiont cells of different sizes show different metabolic profiles. Metaproteomic analysis and statistical evaluation using clustering and random forests, supported by microscopy and flow cytometry, strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: Small symbionts actively divide and may establish cellular symbiont-host interaction, as indicated by highest abundance of the cell division key protein FtsZ and highly abundant chaperones and porins in this initial phase. Large symbionts, on the other hand, apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Highest abundance of enzymes for CO2 fixation, carbon storage and biosynthesis in large symbionts indicates that in this late differentiation stage the symbiont’s metabolism is efficiently geared towards the production of organic material. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.

Like eukaryotes, different bacterial species express one or more Ser/Thr kinases and phosphatases that operate in various signaling networks by catalyzing phosphorylation and dephosphorylation of proteins that can immediately regulate biochemical pathways by altering protein function. The human pathogen Streptococcus pneumoniae encodes a single Ser/Thr kinase-phosphatase couple known as StkP-PhpP, which has shown to be crucial in the regulation of cell wall synthesis and cell division. In this study, we applied proteomics to further understand the physiological role of pneumococcal PhpP and StkP with an emphasis on phosphorylation events on Ser and Thr residues. Therefore, the proteome of the non-encapsulated D39 strain (WT), a kinase (ΔstkP), and phosphatase mutant (ΔphpP) were compared in a mass spectrometry based label-free quantification experiment. Results show that a loss of function of PhpP causes an increased abundance of proteins in the phosphate uptake system Pst. Quantitative proteomic data demonstrated an effect of StkP and PhpP on the two-component systems ComDE, LiaRS, CiaRH, and VicRK. To obtain further information on the function, targets and target sites of PhpP and StkP we combined the advantages of phosphopeptide enrichment using titanium dioxide and spectral library based data evaluation for sensitive detection of changes in the phosphoproteome of the wild type and the mutant strains. According to the role of StkP in cell division we identified several proteins involved in cell wall synthesis and cell division that are apparently phosphorylated by StkP. Unlike StkP, the physiological function of the co-expressed PhpP is poorly understood. For the first time we were able to provide a list of previously unknown putative targets of PhpP. Under these new putative targets of PhpP are, among others, five proteins with direct involvement in cell division (DivIVA, GpsB) and peptidoglycan biosynthesis (MltG, MreC, MacP).

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite for pneumococcal transmission and disease. Current vaccines protect only against disease and colonization caused by a limited number of serotypes, consequently allowing serotype replacement and transmission. Therefore, the development of a broadly protective vaccine against colonization, transmission and disease is desired but requires a better understanding of pneumococcal adaptation to its natural niche. Hence, we measured the levels of free and protein-bound transition metals in human nasal fluid, to determine the effect of metal concentrations on the growth and proteome of S. pneumoniae. Pneumococci cultured in medium containing metal levels comparable to nasal fluid showed a highly distinct proteomic profile compared to standard culture conditions, including the increased abundance of nine conserved, putative surface-exposed proteins. AliA, an oligopeptide binding protein, was identified as the strongest protective antigen, demonstrated by the significantly reduced bacterial load in a murine colonization and a lethal mouse pneumonia model, highlighting its potential as vaccine antigen.

Proteome analyses are often hampered by the low amount of available starting material like a low bacterial cell number obtained from in vivo settings. Here, the single pot solid-phase enhanced sample preparation (SP3) protocol is adapted and combined with effective cell disruption using detergents for the proteome analysis of bacteria available in limited numbers only. Using this optimized protocol, identification of peptides and proteins for different Gram-positive and Gram-negative species can be dramatically increased and, reliable quantification can also be ensured. This adapted method is compared to already established strain-specific sample processing protocols for Staphylococcus aureus, Streptococcus suis, and Legionella pneumophila. The highest species-specific increase in identifications is observed using the adapted method with L. pneumophila samples by increasing protein and peptide identifications up to 300% and 620%, respectively. This increase is accompanied by an improvement in reproducibility of protein quantification and data completeness between replicates. Thus, this protocol is of interest for performing comprehensive proteomics analyses of low bacterial cell numbers from different settings ranging from infection assays to environmental samples.

Bacterial alternative sigma factors are mostly regulated by a partner-switching mechanism. Regulation of the virulence-associated alternative sigma factor SigF of Mycobacterium tuberculosis has been an area of intrigue, with SigF having more predicted regulators than other sigma factors in this organism. Rv1364c is one such predicted regulator, the mechanism of which is confounded by the presence of both anti-sigma factor and anti-sigma factor antagonist functions in a single polypeptide. Using protein binding and phosphorylation assays, we demonstrate that the anti-sigma factor domain of Rv1364c mediates autophosphorylation of its antagonist domain and binds efficiently to SigF. Furthermore, we identified a direct role for the osmosensor serine/threonine kinase PknD in regulating the SigF-Rv1364c interaction, adding to the current understanding about the intersection of these discrete signaling networks. Phosphorylation of SigF also showed functional implications in its DNA binding ability, which may help in activation of the regulon. In M. tuberculosis, osmotic stress-dependent induction of espA, a SigF target involved in maintaining cell wall integrity, is curtailed upon overexpression of Rv1364c, showing its role as an anti-SigF factor. Overexpression of Rv1364c led to induction of another target, pks6, involved in lipid metabolism. This induction was, however, curtailed in the presence of osmotic stress conditions, suggesting modulation of SigF target gene expression via Rv1364c. These data provide evidence that Rv1364c acts an independent SigF regulator that is sensitive to the osmosensory signal, mediating the cross talk of PknD with the SigF regulon. IMPORTANCEMycobacterium tuberculosis, capable of latently infecting the host and causing aggressive tissue damage during active tuberculosis, is endowed with a complex regulatory capacity built of several sigma factors, protein kinases, and phosphatases. These proteins regulate expression of genes that allow the bacteria to adapt to various host-derived stresses, like nutrient starvation, acidic pH, and hypoxia. The cross talk between these systems is not well understood. SigF is one such regulator of gene expression that helps M. tuberculosis to adapt to stresses and imparts virulence. This work provides evidence for its inhibition by the multidomain regulator Rv1364c and activation by the kinase PknD. The coexistence of negative and positive regulators of SigF in pathogenic bacteria reveals an underlying requirement for tight control of virulence factor expression.

Enclosure experiments are frequently used to investigate the impact of changing environmental conditions on microbial assemblages. Yet, how the incuba- tion itself challenges complex bacterial communities is thus far unknown. In this study, metaproteomic profiling, 16S rRNA gene analyses, and cell counts were com- bined to evaluate bacterial communities derived from marine, mesohaline, and oli- gohaline conditions after long-term batch incubations. Early in the experiment, the three bacterial communities were highly diverse and differed significantly in their compositions. Manipulation of the enclosures with terrigenous dissolved organic car- bon resulted in notable differences compared to the control enclosures at this early phase of the experiment. However, after 55 days, bacterial communities in the ma- nipulated and the control enclosures under marine and mesohaline conditions were all dominated by gammaproteobacterium Spongiibacter. In the oligohaline enclo- sures, actinobacterial cluster I of the hgc group (hgc-I) remained abundant in the late phase of the incubation. Metaproteome analyses suggested that the ability to use outer membrane-based internal energy stores, in addition to the previously de- scribed grazing resistance, may enable the gammaproteobacterium Spongiibacter to prevail in long-time incubations. Under oligohaline conditions, the utilization of ex- ternal recalcitrant carbon appeared to be more important (hgc-I). Enclosure experi- ments with complex natural microbial communities are important tools to investi- gate the effects of manipulations. However, species-specific properties, such as individual carbon storage strategies, can cause manipulation-independent effects and need to be considered when interpreting results from enclosures. This study was financially supported by the SAW-funded ATKiM project, which provided funds to D. P. R. Herlemann, C. Meeske, K. Jürgens, S. Markert, and T. Schweder. D. P. R. Herlemann was also supported by the European Regional Develop- ment Fund/Estonian Research Council funded Mobilitas Plus Top Researcher grant MOBTT24. We thank the crew and captain of the RV Meteor (M86, M87) for support during the research cruise. The computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at the PDC Centre for High Performance Computing (PDC-HPC) and Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX). We thank Jana Matulla for excellent technical assis- tance and Stephan Fuchs for his help and advice in MS database construction. We also thank Stefan E. Heiden for valuable help with the CDD BLAST analyses. This study was financially supported by the SAW-funded ATKiM project, which provided funds to D. P. R. Herlemann, C. Meeske, K. Jürgens, S. Markert, and T. Schweder. D. P. R. Herlemann was also supported by the European Regional Develop- ment Fund/Estonian Research Council funded Mobilitas Plus Top Researcher grant MOBTT24. We thank the crew and captain of the RV Meteor (M86, M87) for support during the research cruise. The computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at the PDC Centre for High Performance Computing (PDC-HPC) and Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX). We thank Jana Matulla for excellent technical assis- tance and Stephan Fuchs for his help and advice in MS database construction. We also thank Stefan E. Heiden for valuable help with the CDD BLAST analyses.