To investigate the physiology of microbial mutant strains, they are usually cultivated on solid medium or in batch cultures. Under both conditions, individual nutrients will gradually become growth limiting, and cause changes in the actual specific growth rates, that in turn may profoundly influence the metabolism and consequently the physiology of the cell. While this fact has been well considered in studies of yeasts and bacteria, much fewer reports are available for the applications of methods that take the specific growth rate into consideration for filamentous fungal research [17–19, 25–28].
Because of this similarity to methyltransferases and its localization to the nucleus, LaeA/LAE1 has been postulated to regulate transcription by lysine or arginine protein methyltransferase functions [1, 29]. However, subsequent work showed that LAE1-modulated expression of genes in T. reesei did not correlate with corresponding changes in the histone methylation state at these gene loci . In addition, Patananan et al. , using several different experimental approaches failed to identify a protein or other substrate in A. nidulans that becomes methylated by LaeA. Instead, LaeA was shown to methylate itself at M207, yet this methylation was not essential for LaeA function, and the corresponding M207 does also not occur in the T. reesei LAE1. All this evidence suggests that LaeA/LAE1 may exert its function by binding to other proteins rather than by methylating histones or other DNA-binding proteins.
LaeA has been isolated because of its role as a regulator of secondary metabolism in A. nidulans, and it was therefore surprising that we recently found only a single NRPS to be downregulated in the Δlae1 mutant . In the present study, two PKS and one NRPS genes were affected by lae1 loss-of function, and this effect occurred only at the high growth rate (or was much stronger in the case of the NRPS). The fact that our previous study was performed with lactose grown cells  may explain this discrepancy, because growth on lactose is characterized by a very slow growth rate. Thus the effect of LAE1 on secondary metabolism in T. reesei appears to be growth rate dependent, and more pronounced at the high growth rate. Veiga et al.  have also found a growth rate-dependency of LaeA and VeA function in Penicillium chrysogenum, although in the opposite direction.
Our data also showed that only a comparatively small percentage of the genes that are affected by LAE1 modulation (i.e. 10%) are in fact clustered in the genome. This value is much smaller than clustering percentages obtained for gene expression in T. reesei under other conditions such as conidiation . While a similar clustering analysis of differently expressed genes in T. reesei growing on glycerol and glucose yielded still a lower value (4.3%; C.P.K., unpublished data), the significance of the 10% found in this study is unclear. In any case, the data show that most of the genes that are affected by LAE1 loss of function do not appear to be clustered in the genome of T. reesei.
However, the present study revealed a new level at which LaeA/LAE1 may control gene expression: we show that the expression of 50% of the GCN5-N-acetyltransferases (GNATs) present in the T. reesei genome is altered in the Δlae1 strain, most of them being downregulated. This effect of LAE1 has not been revealed in previous studies , probably due to the use of batch cultures or of different carbon sources. GNATs catalyze the transfer of the acetyl group from acetyl coenzyme A to a primary amine. While – despite of the similarity to the GCN5 protein of the SAGA complex  - some of them may thus fulfill metabolic functions, a subgroup of them is known to acetylate histones at specific lysine residues, a process that leads to transcriptional activation and has been implicated in chromatin assembly [32, 33]. Unfortunately, none of the T. reesei GNATs has so far been functionally investigated, but one – Trire2:120120 – has been shown to be induced on the cellulase inducing carbon sources cellulose [34, 35] and lactose , is downregulated in T. reesei Δlae1 strain  and its overexpression in T. reesei leads to a twofold stimulation of cellulase production. This gene is, however, not expressed on glucose and was therefore not detected under the conditions of this work. However, the above noted correlation between LAE1 function and action of the GNAT Trire2:120120 may also be valid for several of the GNATs detected to be LAE1-dependent in this work. In A. nidulans, LaeA function has recently also been linked to histone acetylation: in a multicopy suppressor screen for genes capable of restoring secondary metabolite production in an A. nidulans ΔlaeA mutant, the histone acetyltransferase EsaA was identified . However, EsaA binds to the target histone by a chromo-domain and is a member of the Myb_Cef protein family (Pfam 11831), and not a GNAT. Thus, a direct link between GNATs and LaeA/LAE1, which may explain the postulated LaeA-dependent chromatin modification , still requires scientific testing. Interestingly, an orthologue of S. cerevisiae SPT10 – an acetyltransferase that directly activates the transcription of histone genes, and whose function is essential for normal cell division  – is also strongly downregulated in the T. reesei Δlae1 mutant. Its downregulation in T. reesei Δlae1 may be a factor contributing to the slower growth observed in this mutant. We should also like to note that the other observed effects such as changes in the growth-rate dependent regulation in the Δlae1 strain would be fully compatible with an action of LAE1 via GNATs, because acetylation by them can also lead to repression of gene expression .
Another group of genes that were significantly influenced by LAE1 in this study – but not detected to be affected previously  – were amino acid transporters. These transporters can reliably be predicted by the presence of a common structural motif consisting of 12 alpha-helical putative transmembrane segments and cytoplasmically located N- and C-terminal hydrophilic regions, and belong to the amino acid/polyamine organocation superfamily . In yeasts, they are usually absent during growth on an inorganic nitrogen source but upregulated once organic nitrogen becomes available, using transcriptional  and/or posttranscriptional mechanisms . In contrast, the present study shows that these amino acid transporters are expressed in T. reesei during growth on an inorganic nitrogen source. These finding is also consistent with the observation that T. reesei prefers amino acids as carbon sources when grown in the presence of cellulose or lactose and amino acid mixtures , and thus regulation of expression of these permease genes still deserves attention. Interestingly, there is now emerging evidence that amino acid uptake in yeast is regulated by GNAT-dependent histone acetylation [44, 45], which would fit to the above supposed role of LAE1 in histone acetylation. We also noted that the effect of LAE1 on amino acid permeases is paralleled by a severe downregulation of a significant number of genes involved in amino acid metabolism in T. reesei Δlae1 (mainly at the high growth rate), and the above findings of regulation of amino acid uptake by LAE1 can therefore be extended to a general effect on amino acid metabolism. Also, some extracellular proteases and oligopeptide transporters were effected by LAE1 (cf. Additional file 1: Table S1), but these two groups as a whole remained not significantly affected.
Most of the other FunCat groups found to be influenced by LAE1 function were already identified in our earlier study, which used lactose as a carbon source to induce cellulase gene expression . Yet differences in the numbers of genes in the individual groups were noted. It is unclear, however, whether these observations are growth rate- or carbon source-specific.