Of the 473 genes in the genome of the bacterium with the smallest genome generated to date, 149 genes have unknown function, emphasising a universal problem; less than 1% of proteins have experimentally determined annotations. Here, we combine the results from state-of-the-art in silico methods for functional annotation and assign functions to 66 of the 149 proteins. Proteins that are still not annotated lack orthologues, lack protein domains, and/ or are membrane proteins. Twenty-four likely transporter proteins are identified indicating the importance of nutrient uptake into and waste disposal out of the minimal bacterial cell in a nutrient-rich environment after removal of metabolic enzymes. Hence, the environment shapes the nature of a minimal genome. Our findings also show that the combination of multiple different state-of-the-art in silico methods for annotating proteins is able to predict functions, even for difficult to characterise proteins and identify crucial gaps for further development.