BACKGROUND
aromatic compounds are widely distributed and abundant constituents of natural organic matter  <cit> , and support as energy-rich substrates the growth of heterotrophic bacteria.
in the environment, aromatic compounds typically co-occur with various and often more easily degradable substrates .
despite the high diversity of potential carbon sources, their total concentration generally does not exceed the μm range.
these low concentrations cause microorganisms to simultaneously utilize multiple different substrates  <cit> .
however, higher  substrate concentrations are expected to occur due to transient pulses  or continuous release .
laboratory experiments with batch cultures of microorganisms often show biphasic  growth with hierarchical utilization of substrates.
firstly, the preferred substrate is exclusively and completely consumed during the first active growth phase.
then, utilization of the subordinate substrate starts following an intermediarily occuring diauxic lag phase  <cit> .
the mechanisms underlying substrate utilization preferences were termed carbon catabolite repression  and have been studied intensively for carbohydrates in a limited number of bacteria, and involve different phosphotransferase systems  for sugar uptake  <cit> .
the pts-independent sugar uptake in pseudomonas spp.
<cit>  and the inability of many nutritional specialists  to utilize carbohydrates, implies the existence of control mechanisms that deviate from the known concepts established with well-studied escherichia coli and bacillus subtilis.
in bacteria able to anaerobically degrade aromatic compounds, few studies have investigated substrate preferences thus far.
under nitrate-reducing conditions and growth at comparable rates, succinate-adapted azoarcus sp.
strain cib preferred utilization of succinate from a binary mixture with benzoate  <cit> , which is mediated by the regulatory protein accr  <cit> .
in contrast to azoarcus sp.
strain cib, related “aromatoleum aromaticum” ebn <dig> preferred benzoate over succinate by repressing succinate uptake via the c4-dicarboxylate trap transporter dctpqm  <cit> .
the denitrifying alphaproteobacterium magnetospirillum sp.
strain pmbn <dig> is the only known isolate utilizing 4-methylbenzoate under anoxic conditions  <cit> .
4-methylbenzoate and benzoate are degraded anaerobically via two distinct, yet analogous pathways  <cit>  that involve:  substrate-coa ligation,  reductive dearomatization of benzoyl-coa by dedicated 4-methylbenzoyl-coa  or benzoyl-coa  reductase, and  a series of β-oxidation-like reactions to yield 3-hydroxy-5-methylpimelyl-coa  or 3-hydroxypimelyl-coa .
further degradation of the latter two intermediates to acetyl-coa then proceeds differently either via reactions analogous to the leucine/isovalerate pathway or by β-oxidation, respectively  <cit> .
in the environment, 4-methylbenzoate and benzoate were reported to co-occur in wastewaters from terephthalic acid production plants at concentrations ranging from  <dig>  to  <dig>  mm  <cit> .
this study combines physiology with differential transcript and protein analyses to investigate the substrate preferences of magnetospirillum sp.
strain pmbn <dig> during anaerobic growth with binary and ternary mixtures of 4-methylbenzoate, benzoate and succinate.
RESULTS
substrate utilization preferences of magnetospirillum sp.
strain pmbn1
binary substrate mixtures.
succinate-adapted cells of magnetospirillum sp.
strain pmbn <dig> growing with a mixture of succinate and benzoate displayed diauxic growth .
in the first active growth phase , the cells preferentially and completely consumed benzoate, while already part of the supplied succinate  was slowly depleted from the medium.
following a  <dig>  h long diauxic lag phase , succinate was rapidly utilized in the second active growth phase .
in contrast to benzoate, other tested co-substrates  were co-utilized with succinate  or succinate was preferred  .
benzoate was also preferentially utilized  from binary mixtures containing the adaptation substrates fumarate, l-malate, oxaloacetate or pyruvate .
in analogy to succinate, also here the adaptation substrates were already partially and slowly depleted during benzoate utilization with the duration of diauxic lag phases ranging from  <dig>  to  <dig>  h. only in combination with acetate as adaptation substrate, monophasic growth with co-utilization of benzoate was observed.
in contrast, when supplying 4-methylbenzoate as co-substrate, diauxic growth with exclusive and preferential consumption of acetate was observed , whereas pyruvate and 4-methylbenzoate were co-utilized  .
using cells adapted to 4-methylbenzoate, both tested aromatic co-substrates  were exclusively and completely utilized during phase  <dig>  followed by a 4–5 h long diauxic lag phase and utilization of 4-methylbenzoate only during phase  <dig> .figure  <dig>
anaerobic growth of
magnetospirillum
sp.
strain pmbn <dig> with binary and ternary substrate mixtures of 4-methylbenzoate, benzoate and succinate.
growth behavior and substrate utilization profiles of magnetospirillum sp.
strain pmbn <dig> adapted to succinate  or 4-methylbenzoate .
anaerobic growth of
magnetospirillum
sp.
strain pmbn <dig> with binary mixtures of aliphatic and aromatic acids
binary mixture of adaptation and co-substrate
phase 1
phase 2
phase 3
adaptation substrate
co-substrate
od
max
μ
max
duration of diauxic lag phase
od
max
μ
max
cells adapted to succinate
benzoate
succinate
cells adapted to 4-methylbenzoate
benzoate
phenylacetate
cells adapted to other aliphatic acids
acetate
benzoate
benzoate
benzoate
benzoate
cells adapted to other aromatic acids
succinate
preferentially utilized substrates are highlighted in boldface.
applied substrate concentrations are indicated in parentheses.
values for maximal optical density  and maximum specific growth rates  are based on three replicate cultures with standard deviations of below 5%.
μmax was calculated from the slope of the active growth phase  according to μmax = m × 1/Δod.
in the case of diauxic growth, phase  <dig> corresponds to the first active growth phase.
in the case of monophasic growth, phase  <dig> corresponds to the only observed active growth phase.
if present, phase  <dig> corresponds to a second active growth phase, with phase  <dig> representing the diauxic lag phase between phases  <dig> and  <dig> .
for further details see additional file 1: figures s1–s <dig>
ternary substrate mixture.
cells adapted to 4-methylbenzoate were shifted to a mixture of 4-methylbenzoate, benzoate and succinate, resulting in diauxic growth with preferential and complete utilization of benzoate during phase  <dig> .
again, this was accompanied by partial  depletion of the supplied succinate.
following a short  diauxic lag phase, 4-methylbenzoate and the bulk of succinate were co-utilized in phase  <dig>  the same growth behavior and substrate preference was observed, if succinate-adapted cells were used as inoculum .
a notable observation is the unaltered utilization hierarchy with the ternary mixture, i.e.
benzoate over succinate and 4-methylbenzoate, mirroring the preferences observed with respective binary mixtures .table  <dig>
anaerobic growth of
magnetospirillum
sp.
strain pmbn <dig> with a ternary mixture of 4-methylbenzoate, benzoate and succinate
ternary mixture of adaptation and co-substrates
phase 1
phase 2
phase 3
adaptation substrate
co-substrate 1
co-substrate 2
od
max
μ
max
duration of diauxic lag phase
od
max
μ
max
benzoate
for details see description to table  <dig>
transcript and protein dynamics
time-resolved abundance profiles of transcripts  and proteins  related to the uptake and catabolism of 4-methylbenzoate, benzoate and succinate were determined during anaerobic diauxic growth with the ternary substrate mixture, and related to that of succinate-grown cells as reference .
for comparison, the same transcripts/proteins were profiled accordingly at lower time resolution  for respective binary substrate mixtures  and single substrates.figure  <dig>
time-resolved profiles of selected transcripts and corresponding proteins  related to uptake and catabolism of benzoate , 4-methylbenzoate  and succinate .
4-methylbenzoate-adapted cells of magnetospirillum sp.
strain pmbn <dig> were shifted to a ternary substrate mixture  of 4-methylbenzoate , benzoate  and succinate .
abbreviations for experiments with single substrates: m, 4-methylbenzoate; b, benzoate.
the fold change in protein and ratio of transcript abundance were determined by comparison to succinate-adapted cells as reference.
see legend to figure  <dig> for name and predicted functions of selected transcripts/proteins.
for detailed information see additional file 1: tables s <dig> and s <dig>
fold change in the abundance of proteins related to uptake and catabolism of benzoate , 4-methylbenzoate  and succinate  during anaerobic growth of
magnetospirillum
sp.
strain pmbn <dig> with substrate mixtures or single substrates .
sampling points for proteomic analyses are marked in green in the growth curves displayed on the left panel.
identified proteins are marked in blue.
for further details  see additional file 1: table s <dig>  *, transcript profiles of selected genes during growth with the ternary substrate mixture are shown in figure  <dig>  protein names :  aconitase  <dig>   benzoate-coa ligase,  benzoyl-coa reductase,  benzoate/h+ symporter,  cyclohex- <dig> -diene-1-carbonyl-coa hydratase,  4-methylcyclohex- <dig> -diene-1-carbonyl-coa hydratase,  c4-dicarboxylate/na+ symporter,  trap-type c4-dicarboxylate uptake transporter,  fumarate hydratase,  citrate synthase,  6-hydroxycyclohex-1-ene-1-carbonyl-coa dehydrogenase,  6-hydroxy-4-methylcyclohex-1-ene-1-carbonyl-coa dehydrogenase,  isocitrate dehydrogenase,  2-oxoglutarate:ferredoxin oxidoreductase,  nadp+-dependent malic enzyme,  4-methylbenzoyl-coa reductase,  4-methylbenzoate-coa ligase,  malate dehydrogenase,  6-oxocyclohex-1-ene-1-carbonyl-coa hydrolase,  4-methyl-6-oxocyclohex-1-ene-1-carbonyl-coa hydrolase,  predicted abc-type 4-methylbenzoate uptake transporter,  pyruvate dehydrogenase complex,  succinate dehydrogenase,  2-oxoglutarate dehydrogenase complex,  succinyl-coa ligase.
compound names:  <dig>  benzoate;  <dig>  benzoyl-coa;  <dig>  cyclohex- <dig> -diene-carbonyl-coa;  <dig>  4-methylbenzoate;  <dig>  4-methylbenzoyl-coa;  <dig>  4-methylcyclohex- <dig> -diene-1-carbonyl-coa;  <dig>  succinate.
benzoate uptake and catabolism .
benzoate utilization during phase  <dig> coincided with the largest abundance increase  for the protein constituents of the anaerobic benzoyl-coa pathway , which then constantly decreased during phases  <dig> and  <dig> .
immediately  after complete depletion of benzoate from the medium, the transcript level of the c-subunit of benzoyl-coa reductase  decreased markedly  and reached its minimum  at the end of the diauxic lag phase.
transcripts of benzoate-coa ligase  behaved similarly, but the overall abundance change was far less pronounced  .
benzoate uptake in magnetospirillum sp.
strain pmbn <dig> is predicted to proceed via a benzoate/h+ symporter homologous to benk  <cit> .
in cells of magnetospirillum sp.
strain pmbn <dig>  benk was detected during growth with benzoate or 4-methylbenzoate, but not with succinate.
in contrast to the catabolic constituents of the anaerobic benzoyl-coa pathway, transcript and protein levels of benk changed only slightly across phases 1– <dig> .
4-methylbenzoate uptake and catabolism .
despite non-utilization of 4-methylbenzoate during phase  <dig> of diauxic growth, four proteins of the anaerobic 4-methylbenzoyl-coa pathway  displayed increased abundances .
at the end of the diauxic lag phase, these proteins markedly increased in abundance, reaching their highest levels during maximal 4-methylbenzoate utilization in phase  <dig> .
transcript levels of predicted 4-methylbenzoate-coa ligase  and of the c-subunit of 4-methylbenzoyl-coa reductase  were initially low , but started to increase at benzoate concentrations < <dig>  mm, which was followed by a strong increase upon benzoate depletion , and likewise reaching maximal levels  in phase  <dig> .
incongruent transcript  and protein  levels of mbrc during phase  <dig> suggest that the highly abundant proteins of 4-methylbenzoate degradation  <cit>  might only be slowly degraded.
at present, the proteomic dataset suggests uptake of 4-methylbenzoate by an abc transporter, most subunits of which  were present in cells grown with 4-methylbenzoate, but absent in cells grown with succinate or benzoate .
the transcript level of orf <dig> immediately increased at the end of phase  <dig> , peaked at the end of the diauxic lag phase , and remained at high levels during phase  <dig>  .
the protein level of orf <dig> changed accordingly and the four membrane-associated components  of the abc transporter were identified only in the membrane protein fraction of cells harvested during phases  <dig> and  <dig> .
succinate uptake and catabolism .
among all identified proteins, the β-subunit  of the e <dig> and the e <dig>  component of pyruvate dehydrogenase  displayed the largest decreases in abundance .
notably, during bulk succinate utilization in phase  <dig>  both proteins attained similarly high levels as observed in the succinate-grown reference .
the changes in transcript levels of pdhb essentially correlated with changes in the pdhb protein level.
in addition to pyruvate dehydrogenase, metabolism of succinate to pyruvate is suggested to also involve nadp+-dependent malic enzyme , which was not detected by 2d dige.
only at benzoate concentrations > <dig>  mm, the transcript level of maeb was slightly  reduced .
the abundance profiles of maeb and pyruvate dehydrogenase agree with their dispensable function during benzoate or 4-methylbenzoate catabolism .
correspondingly, minimal transcript/protein abundances of these two enzymes were observed when the two aromatic substrates were utilized  .
based on the proteomic dataset, uptake of succinate in magnetospirillum sp.
strain pmbn <dig> is suggested to involve two different transporters.
firstly, a c4-dicarboxylate trap transporter, displaying high amino acid sequence identities  to characterized dctpqm from rhodobacter capsulatus  <cit> , and secondly, the c4-dicarboxylate/cation symporter dcta with 63% amino acid sequence identity to characterized dcta from sinorhizobium meliloti  <cit> .
2d dige captured only the periplasmic c4-dicarboxylate-binding protein dctp of the trap transporter, which displayed decreased abundance  during benzoate utilization in phase  <dig>  and increased continuously upon benzoate depletion during phases  <dig> and  <dig> .
dctqm were both identified in the membrane protein-enriched fraction of succinate-grown cells, whereas only one of the two subunits was detected in cells grown with benzoate, or across phases 1– <dig>  symporter dcta was identified in succinate-grown cells and across phases 1– <dig>  but not in cells grown with benzoate or 4-methylbenzoate as single substrate .
transcript levels of dctp, dctm and dcta were similar across phases 1– <dig>  the extent and dynamics of which were comparable to that of the maeb transcript .
minimal transcript levels of dctp, dctm and dcta were observed during growth with benzoate or 4-methylbenzoate as single substrate .
these transcript profiles are in accord with the known c4-dicarboxylate-dependent activation of dctpqm and dcta expression by dedicated c4-dicarboxylate-responsive two-component sensory/regulatory systems as known from r. capsulatus  and rhizobia   <cit> .
homologues of those are also encoded in the genome of magnetospirillum sp.
strain pmbn <dig> .
ternaryversusbinary substrate mixtures .
protein profiles of cells grown with selected binary mixtures  were compared to those from cultures grown with the ternary substrate mixture.
overall, the dynamics and extent of protein abundance changes matched those observed with the ternary substrate mixture.
however, differences were observed for some proteins :  the protein constituents of the anaerobic benzoyl-coa pathway were generally higher in abundance, if 4-methylbenzoate was additonally present.
in the absence of benzoate, dctp and pdhb levels were unaffected by 4-methylbenzoate, agreeing with co-utilization of succinate and 4-methylbenzoate .
however, if provided together, 4-methylbenzoate and benzoate had an additive negative effect on these two proteins, which could contribute to the observed decreased succinate depletion during phase  <dig> with the ternary substrate mixture .
it should be noted, however, that the observed differences might be related to using either succinate or 4-methylbenzoate as adaptation substrate in the compared experiments.
the abundance change of dctp and pdhb was incongruent in the presence of succinate .
i.e., dctp levels increased from − <dig> - to − <dig> -fold in phase  <dig>  while pdhb levels remained minimal at − <dig> -fold, thus indicating that unlike dctp, repression of pdhb is unaffected by the co-substrate succinate.
discussion
benzoate is preferred by magnetospirillum sp.
strain pmbn1
magnetospirillum sp.
strain pmbn <dig> is currently the only described isolate capable of anaerobically degrading 4-methylbenzoate  <cit> .
anaerobic degradation of 4-methylbenzoate and benzoate proceeds via two analogous pathways each requiring a distinct set of enzymes  <cit> .
despite adaptation to 4-methylbenzoate and thereby “transferred” initial presence of catabolic proteins for 4-methylbenzoate degradation, magnetospirillum sp.
strain pmbn <dig> preferred utilization of benzoate from mixtures with 4-methylbenzoate .
this preference cannot be attributed to markedly different growth rates  .
however, lower growth yields − <dig> for benzoate) despite the higher free energies  obtained from complete oxidation of 4-methylbenzoate   <cit> , indicate less efficient metabolism with 4-methylbenzoate.
in addition, the range of substrates feeding into the 4-methylbenzoyl-coa degradation pathway is to our present knowledge limited to 4-methylbenzoate, whereas from the aromatic substrates of magnetospirillum sp.
strain pmbn <dig>  <cit> , at least  <dig>  are known from other denitrifying bacteria to be channeled into the central anaerobic benzoyl-coa pathway .
indeed, also phenylacetate repressed 4-methylbenzoate utilization in magnetospirillum sp.
strain pmbn <dig> .
one may speculate that benzoate preference of magnetospirillum sp.
strain pmbn <dig> reflects the higher probability to encounter substrates of the anaerobic benzoyl-coa pathway in the environment.
in thauera aromatica ar- <dig>  benzoate repressed anaerobic  <dig> -dihydroxybenzoate degradation, which is apparently degraded via a catabolic route distinct from the anaerobic benzoyl-coa pathway  <cit> .
it seems thus likely that the anaerobic benzoyl-coa pathway might generally be preferred over more specialized  aromatic degradation pathways.
aerobic and anaerobic aromatic compound degrading bacteria in most reported cases prefer succinate over aromatic acids  <cit> .
the opposite was described recently for the first time in the denitrifying betaproteobacterium “a.
aromaticum” ebn <dig>  prefering benzoate despite adaptation to anaerobic growth with succinate  <cit> .
in this study, the same unusual preference was observed in the alphaproteobacterium magnetospirillum sp.
strain pmbn <dig>  which is phylogenetically distinct from “a.
aromaticum” ebn <dig>  in addition to succinate, growth behavior and substrate preferences of both strains were similar with mixtures of benzoate and the adaptation substrates fumarate or malate  and acetate , but differed for oxaloacetate and pyruvate.
while “a.
aromaticum” ebn <dig> grew monophasically with one of the latter and benzoate, diauxic growth with benzoate preference was observed in magnetospirillum sp.
strain pmbn <dig>  substrate preferences in both strains could not be attributed to differences in growth rate    and complexity of degradation pathways.
taken together, magnetospirillum sp.
strain pmbn <dig> and “a.
aromaticum” ebn <dig> seem to be highly specialized on utilization of aromatic acids  under anoxic conditions, since aliphatic substrates  were first and exclusively utilized only from two  tested binary substrate mixtures.
benzoate mediates repression of 4-methylbenzoate uptake
4-methylbenzoate-specific detection of a putative abc transporter for 4-methylbenzoate uptake  and the strong increase in transcript levels of its periplasmic solute-binding protein  upon benzoate depletion , suggest benzoate to mediate repression of 4-methylbenzoate uptake.
this is supported by non-utilization of 4-methylbenzoate during phase  <dig>  despite presence of some catabolic proteins  .
expression of genes for anaerobic catabolism of aromatic acids is typically controlled by regulatory proteins recognizing corresponding aryl-coa esters .
inhibition of 4-methylbenzoate uptake would prevent its intracellular presence and thus generation of 4-methylbenzoyl-coa as putative inductor for expression of genes for 4-methylbenzoate catabolism.
this is evident, e.g., from observed minimal transcript levels of mcla, encoding 4-methylbenzoate-coa ligase, and mbrc during phase  <dig>  it is known that benzoate-coa ligases of azoarcus sp.
strain cib  <cit> , t. aromatica k <dig>  <cit>  and magnetospirillum sp.
strain ts- <dig>  <cit>  also convert 4-fluorobenzoate at high rates , and 3-methylbenzoate-coa ligase  of azoarcus sp.
strain cib exhibits the same activity with 3-methylbenzoate and benzoate  <cit> .
the high  amino acid sequence identities of benzoate-coa ligase  of magnetospirillum sp.
strain pmbn <dig> and mbda of azoarcus sp.
strain cib may suggest bcla of magnetospirillum sp.
strain pmbn <dig> to possibly also accept 4-methylbenzoate as substrate.
in this case, excluding 4-methylbenzoate from entering the cell would most effectively prevent induction of the 4-methylbenzoyl-coa pathway.
benzoate mediates a dual repression of succinate uptake and utilization
in case of “a.
aromaticum” ebn <dig>  it was assumed that benzoate mediates complete repression of succinate uptake by negatively controlling dctsr-dependent activation of dctpqm expression  <cit> .
despite the similar diauxic growth behavior and presence of homologous dctpqm/dctsr , magnetospirillum sp.
strain pmbn <dig> partially depleted succinate already during benzoate utilization.
this may be due to presence of an additional transporter for c4-dicarboxylate uptake  together with a cognate c4-dicarboxylate-responsive two-component sensory/regulatory system  in magnetospirillum sp.
strain pmbn <dig>  simultaneous employment of dcta and dctpqm in succinate uptake has been reported for pseudomonas aeruginosa pao <dig>  where dctbd  controls the coordinate expression of dcta and dctpqm  <cit> .
in magnetospirillum sp.
strain pmbn <dig>  succinate-specific detection  of dcta and dctpqm indeed suggests participation of both transporters in succinate uptake.
the coding genes are apparently coordinately expressed, considering the similar dynamics in their transcript levels across phases 1– <dig> .
the only slightly reduced levels of c4-dicarboxylate transporter transcripts during phase  <dig>  suggest that benzoate does not have a similarly strong negative effect on dctsr- and/or dctbd-dependent transcriptional activation of dctpqm/dcta expression in magnetospirillum sp.
strain pmbn <dig>  compared to “a.
aromaticum” ebn <dig>  together with the partial depletion of succinate already during phase  <dig>  repression of succinate uptake cannot be the only determinant for the observed diauxie in magnetospirillum sp.
strain pmbn <dig>
succinate catabolism involves reactions of the tca cycle, as well as malic enzyme  and pyruvate dehydrogenase  to generate acetyl-coa.
reduced transcript/protein levels of the latter two enzymes in magnetospirillum sp.
strain pmbn <dig> during benzoate utilization in phase  <dig> , suggest benzoate to also mediate inhibition of succinate conversion to acetyl-coa.
the abundance decrease of the pdhb transcript, which was most pronounced and prolonged until complete benzoate depletion , indicates that benzoate could mediate transcriptional repression of pyruvate dehydrogenase as main target for inhibition of succinate catabolism.
since expression of pyruvate dehydrogenase genes requires intracellular pyruvate as inductor in s. meliloti  <cit>  and e. coli  <cit> , one may speculate that due to impeded succinate uptake together with reduced maeb-dependent pyruvate generation, intracellular pyruvate levels are insufficient for activation of pyruvate dehydrogenase gene expression.
the partial depletion  of pyruvate during preferred benzoate utilization  could suggest a more direct involvement of benzoate in repressing the expression of pyruvate dehydrogenase genes.
apparently, also here full expression of the pyruvate dehydrogenase genes as basis for maximal pyruvate consumption could only be achieved upon complete depletion of benzoate.
uptake of succinate, l-malate and fumarate in magnetospirillum sp.
strain pmbn <dig> most likely proceeds via dctpqm and dcta, whereas these transporters are generally not involved in import of pyruvate, oxaloacetate or acetate  <cit> .
with the exception of acetate, pyruvate dehydrogenase is essential for intracellular conversion of these five aliphatic acids to acetyl-coa.
co-utilization of acetate from a mixture with benzoate thus agrees with the importance of pyruvate dehydrogenase in achieving preferential benzoate utilization in magnetospirillum sp.
strain pmbn <dig>
possible regulatory mechanisms for simultaneous repression of 4-methylbenzoate and succinate utilization
at present we can only speculate about the mechanism underlying benzoate-mediated repression of 4-methylbenzoate and succinate utilization in magnetospirillum sp.
strain pmbn <dig>  in case of 4-methylbenzoate, a possible regulatory mechanism could involve the predicted tetr-type transcriptional regulator orf <dig>  and benzoyl-coa as the common intermediate of anaerobic benzoate and phenylacetate degradation.
one may speculate that in analogy to the tetr-type repressor dest   <cit> , benzoyl-coa may act as co-repressor of orf <dig>  re-establishing transcriptional repression of genes for 4-methylbenzoate uptake and catabolism in cells that were originally adapted to 4-methylbenzoate .
on the contrary, shared intermediates  occuring in the anaerobic degradation of benzoate, 4-methylbenzoate, acetate, phenylacetate or 4-hydroxybenzoate, suggest benzoate itself as inductor to mediate the dual repression of c4-dicarboxylate uptake and catabolism.
one may speculate that benzoate interferes directly or indirectly with dctsr- and/or dctbd-mediated signal transduction, e.g.
by modulating kinase activities  <cit> .
in addition, benzoate may activate transcriptional repressor  <cit> , facilitate degradation of target transcripts  <cit> , or negatively affect translation of transcripts at the sensory/regulatory, uptake and catabolic level  <cit> .
CONCLUSIONS
next to “a.
aromaticum” ebn <dig>  the only distantly related magnetospirillum sp.
strain pmbn <dig> is the second reported pure culture displaying an unexpected diauxie  with benzoate preference from mixtures with c4-dicarboxylates.
despite the similar growth behavior, apparently different molecular mechanisms govern the sequential utilization in both strains, adding to the so far largely unexplored regulatory potential within anaerobic aromatic compound-degrading bacteria.
preferential utilization of aromatic acids, or their co-utilization with aliphatic acids, seems to be more common among these degradation specialists than previously expected.
co-utilization even at the applied high  concentrations of aromatic and aliphatic acids further suggests only weak or actually absent carbon catabolite control.
the unique nutritional feature of magnetospirillum sp.
strain pmbn <dig> is the capacity to anaerobically grow with 4-methylbenzoate, in addition to aromatic acids degraded via the widespread anaerobic benzoyl-coa pathway.
one may speculate that the observed temporal separation of benzoate and 4-methylbenzoate utilization, and thereby successive operation of the benzoyl-coa and 4-methylbenzoyl-coa pathways allow preventing metabolic inefficiencies, e.g.
by unbalanced draining of shared co-factors or the competitive inhibition of analogous enzymes.

 aromatic compounds are widely distributed and abundant constituents of natural organic matter, and support as energy-rich substrates the growth of heterotrophic bacteria. in the environment, aromatic compounds typically co-occur with various and often more easily degradable substrates. despite the high diversity of potential carbon sources, their total concentration generally does not exceed the μm range. these low concentrations cause microorganisms to simultaneously utilize multiple different substrates. however, higher substrate concentrations are expected to occur due to transient pulses or continuous release. laboratory experiments with batch cultures of microorganisms often show biphasic growth with hierarchical utilization of substrates. firstly, the preferred substrate is exclusively and completely consumed during the first active growth phase. then, utilization of the subordinate substrate starts following an intermediarily occuring diauxic lag phase. the mechanisms underlying substrate utilization preferences were termed carbon catabolite repression and have been studied intensively for carbohydrates in a limited number of bacteria, and involve different phosphotransferase systems for sugar uptake. the pts-independent sugar uptake in pseudomonas spp. and the inability of many nutritional specialists to utilize carbohydrates, implies the existence of control mechanisms that deviate from the known concepts established with well-studied escherichia coli and bacillus subtilis. in bacteria able to anaerobically degrade aromatic compounds, few studies have investigated substrate preferences thus far. under nitrate-reducing conditions and growth at comparable rates, succinate-adapted azoarcus sp. strain cib preferred utilization of succinate from a binary mixture with benzoate, which is mediated by the regulatory protein accr. in contrast to azoarcus sp. strain cib, related “aromatoleum aromaticum” ebn preferred benzoate over succinate by repressing succinate uptake via the c4-dicarboxylate trap transporter dctpqm. the denitrifying alphaproteobacterium magnetospirillum sp. strain pmbn is the only known isolate utilizing 4-methylbenzoate under anoxic conditions. 4-methylbenzoate and benzoate are degraded anaerobically via two distinct, yet analogous pathways that involve: substrate-coa ligation, reductive dearomatization of benzoyl-coa by dedicated 4-methylbenzoyl-coa or benzoyl-coa reductase, and a series of β-oxidation-like reactions to yield 3-hydroxy-5-methylpimelyl-coa or 3-hydroxypimelyl-coa. further degradation of the latter two intermediates to acetyl-coa then proceeds differently either via reactions analogous to the leucine/isovalerate pathway or by β-oxidation, respectively. in the environment, 4-methylbenzoate and benzoate were reported to co-occur in wastewaters from terephthalic acid production plants at concentrations ranging from to  mm. this study combines physiology with differential transcript and protein analyses to investigate the substrate preferences of magnetospirillum sp. strain pmbn during anaerobic growth with binary and ternary mixtures of 4-methylbenzoate, benzoate and succinate.  substrate utilization preferences of magnetospirillum sp. strain pmbn1 binary substrate mixtures. succinate-adapted cells of magnetospirillum sp. strain pmbn growing with a mixture of succinate and benzoate displayed diauxic growth. in the first active growth phase, the cells preferentially and completely consumed benzoate, while already part of the supplied succinate was slowly depleted from the medium. following a  h long diauxic lag phase, succinate was rapidly utilized in the second active growth phase. in contrast to benzoate, other tested co-substrates were co-utilized with succinate or succinate was preferred. benzoate was also preferentially utilized from binary mixtures containing the adaptation substrates fumarate, l-malate, oxaloacetate or pyruvate. in analogy to succinate, also here the adaptation substrates were already partially and slowly depleted during benzoate utilization with the duration of diauxic lag phases ranging from to  h. only in combination with acetate as adaptation substrate, monophasic growth with co-utilization of benzoate was observed. in contrast, when supplying 4-methylbenzoate as co-substrate, diauxic growth with exclusive and preferential consumption of acetate was observed, whereas pyruvate and 4-methylbenzoate were co-utilized. using cells adapted to 4-methylbenzoate, both tested aromatic co-substrates were exclusively and completely utilized during phase followed by a 4–5 h long diauxic lag phase and utilization of 4-methylbenzoate only during phase.figure anaerobic growth of magnetospirillum sp. strain pmbn with binary and ternary substrate mixtures of 4-methylbenzoate, benzoate and succinate. growth behavior and substrate utilization profiles of magnetospirillum sp. strain pmbn adapted to succinate or 4-methylbenzoate. anaerobic growth of magnetospirillum sp. strain pmbn with binary mixtures of aliphatic and aromatic acids binary mixture of adaptation and co-substrate phase 1 phase 2 phase 3 adaptation substrate co-substrate od max μ max duration of diauxic lag phase od max μ max cells adapted to succinate benzoate succinate cells adapted to 4-methylbenzoate benzoate phenylacetate cells adapted to other aliphatic acids acetate benzoate benzoate benzoate benzoate cells adapted to other aromatic acids succinate preferentially utilized substrates are highlighted in boldface. applied substrate concentrations are indicated in parentheses. values for maximal optical density and maximum specific growth rates are based on three replicate cultures with standard deviations of below 5%. μmax was calculated from the slope of the active growth phase according to μmax = m × 1/Δod. in the case of diauxic growth, phase corresponds to the first active growth phase. in the case of monophasic growth, phase corresponds to the only observed active growth phase. if present, phase corresponds to a second active growth phase, with phase representing the diauxic lag phase between phases and. for further details see additional file 1: figures s1–s ternary substrate mixture. cells adapted to 4-methylbenzoate were shifted to a mixture of 4-methylbenzoate, benzoate and succinate, resulting in diauxic growth with preferential and complete utilization of benzoate during phase. again, this was accompanied by partial depletion of the supplied succinate. following a short diauxic lag phase, 4-methylbenzoate and the bulk of succinate were co-utilized in phase the same growth behavior and substrate preference was observed, if succinate-adapted cells were used as inoculum. a notable observation is the unaltered utilization hierarchy with the ternary mixture, i.e. benzoate over succinate and 4-methylbenzoate, mirroring the preferences observed with respective binary mixtures.table anaerobic growth of magnetospirillum sp. strain pmbn with a ternary mixture of 4-methylbenzoate, benzoate and succinate ternary mixture of adaptation and co-substrates phase 1 phase 2 phase 3 adaptation substrate co-substrate 1 co-substrate 2 od max μ max duration of diauxic lag phase od max μ max benzoate for details see description to table  transcript and protein dynamics time-resolved abundance profiles of transcripts and proteins related to the uptake and catabolism of 4-methylbenzoate, benzoate and succinate were determined during anaerobic diauxic growth with the ternary substrate mixture, and related to that of succinate-grown cells as reference. for comparison, the same transcripts/proteins were profiled accordingly at lower time resolution for respective binary substrate mixtures and single substrates.figure time-resolved profiles of selected transcripts and corresponding proteins related to uptake and catabolism of benzoate, 4-methylbenzoate and succinate. 4-methylbenzoate-adapted cells of magnetospirillum sp. strain pmbn were shifted to a ternary substrate mixture of 4-methylbenzoate, benzoate and succinate. abbreviations for experiments with single substrates: m, 4-methylbenzoate; b, benzoate. the fold change in protein and ratio of transcript abundance were determined by comparison to succinate-adapted cells as reference. see legend to figure  for name and predicted functions of selected transcripts/proteins. for detailed information see additional file 1: tables s and s fold change in the abundance of proteins related to uptake and catabolism of benzoate, 4-methylbenzoate and succinate during anaerobic growth of magnetospirillum sp. strain pmbn with substrate mixtures or single substrates. sampling points for proteomic analyses are marked in green in the growth curves displayed on the left panel. identified proteins are marked in blue. for further details see additional file 1: table s *, transcript profiles of selected genes during growth with the ternary substrate mixture are shown in figure  protein names: aconitase benzoate-coa ligase, benzoyl-coa reductase, benzoate/h+ symporter, cyclohex- -diene-1-carbonyl-coa hydratase, 4-methylcyclohex- -diene-1-carbonyl-coa hydratase, c4-dicarboxylate/na+ symporter, trap-type c4-dicarboxylate uptake transporter, fumarate hydratase, citrate synthase, 6-hydroxycyclohex-1-ene-1-carbonyl-coa dehydrogenase, 6-hydroxy-4-methylcyclohex-1-ene-1-carbonyl-coa dehydrogenase, isocitrate dehydrogenase, 2-oxoglutarate:ferredoxin oxidoreductase, nadp+-dependent malic enzyme, 4-methylbenzoyl-coa reductase, 4-methylbenzoate-coa ligase, malate dehydrogenase, 6-oxocyclohex-1-ene-1-carbonyl-coa hydrolase, 4-methyl-6-oxocyclohex-1-ene-1-carbonyl-coa hydrolase, predicted abc-type 4-methylbenzoate uptake transporter, pyruvate dehydrogenase complex, succinate dehydrogenase, 2-oxoglutarate dehydrogenase complex, succinyl-coa ligase. compound names: benzoate; benzoyl-coa; cyclohex- -diene-carbonyl-coa; 4-methylbenzoate; 4-methylbenzoyl-coa; 4-methylcyclohex- -diene-1-carbonyl-coa; succinate. benzoate uptake and catabolism. benzoate utilization during phase coincided with the largest abundance increase for the protein constituents of the anaerobic benzoyl-coa pathway, which then constantly decreased during phases and. immediately after complete depletion of benzoate from the medium, the transcript level of the c-subunit of benzoyl-coa reductase decreased markedly and reached its minimum at the end of the diauxic lag phase. transcripts of benzoate-coa ligase behaved similarly, but the overall abundance change was far less pronounced. benzoate uptake in magnetospirillum sp. strain pmbn is predicted to proceed via a benzoate/h+ symporter homologous to benk. in cells of magnetospirillum sp. strain pmbn benk was detected during growth with benzoate or 4-methylbenzoate, but not with succinate. in contrast to the catabolic constituents of the anaerobic benzoyl-coa pathway, transcript and protein levels of benk changed only slightly across phases 1–. 4-methylbenzoate uptake and catabolism. despite non-utilization of 4-methylbenzoate during phase of diauxic growth, four proteins of the anaerobic 4-methylbenzoyl-coa pathway displayed increased abundances. at the end of the diauxic lag phase, these proteins markedly increased in abundance, reaching their highest levels during maximal 4-methylbenzoate utilization in phase. transcript levels of predicted 4-methylbenzoate-coa ligase and of the c-subunit of 4-methylbenzoyl-coa reductase were initially low, but started to increase at benzoate concentrations <  mm, which was followed by a strong increase upon benzoate depletion, and likewise reaching maximal levels in phase. incongruent transcript and protein levels of mbrc during phase suggest that the highly abundant proteins of 4-methylbenzoate degradation might only be slowly degraded. at present, the proteomic dataset suggests uptake of 4-methylbenzoate by an abc transporter, most subunits of which were present in cells grown with 4-methylbenzoate, but absent in cells grown with succinate or benzoate. the transcript level of orf immediately increased at the end of phase, peaked at the end of the diauxic lag phase, and remained at high levels during phase. the protein level of orf changed accordingly and the four membrane-associated components of the abc transporter were identified only in the membrane protein fraction of cells harvested during phases and. succinate uptake and catabolism. among all identified proteins, the β-subunit of the e and the e component of pyruvate dehydrogenase displayed the largest decreases in abundance. notably, during bulk succinate utilization in phase both proteins attained similarly high levels as observed in the succinate-grown reference. the changes in transcript levels of pdhb essentially correlated with changes in the pdhb protein level. in addition to pyruvate dehydrogenase, metabolism of succinate to pyruvate is suggested to also involve nadp+-dependent malic enzyme, which was not detected by 2d dige. only at benzoate concentrations >  mm, the transcript level of maeb was slightly reduced. the abundance profiles of maeb and pyruvate dehydrogenase agree with their dispensable function during benzoate or 4-methylbenzoate catabolism. correspondingly, minimal transcript/protein abundances of these two enzymes were observed when the two aromatic substrates were utilized. based on the proteomic dataset, uptake of succinate in magnetospirillum sp. strain pmbn is suggested to involve two different transporters. firstly, a c4-dicarboxylate trap transporter, displaying high amino acid sequence identities to characterized dctpqm from rhodobacter capsulatus, and secondly, the c4-dicarboxylate/cation symporter dcta with 63% amino acid sequence identity to characterized dcta from sinorhizobium meliloti. 2d dige captured only the periplasmic c4-dicarboxylate-binding protein dctp of the trap transporter, which displayed decreased abundance during benzoate utilization in phase and increased continuously upon benzoate depletion during phases and. dctqm were both identified in the membrane protein-enriched fraction of succinate-grown cells, whereas only one of the two subunits was detected in cells grown with benzoate, or across phases 1– symporter dcta was identified in succinate-grown cells and across phases 1– but not in cells grown with benzoate or 4-methylbenzoate as single substrate. transcript levels of dctp, dctm and dcta were similar across phases 1– the extent and dynamics of which were comparable to that of the maeb transcript. minimal transcript levels of dctp, dctm and dcta were observed during growth with benzoate or 4-methylbenzoate as single substrate. these transcript profiles are in accord with the known c4-dicarboxylate-dependent activation of dctpqm and dcta expression by dedicated c4-dicarboxylate-responsive two-component sensory/regulatory systems as known from r. capsulatus and rhizobia. homologues of those are also encoded in the genome of magnetospirillum sp. strain pmbn. ternaryversusbinary substrate mixtures. protein profiles of cells grown with selected binary mixtures were compared to those from cultures grown with the ternary substrate mixture. overall, the dynamics and extent of protein abundance changes matched those observed with the ternary substrate mixture. however, differences were observed for some proteins: the protein constituents of the anaerobic benzoyl-coa pathway were generally higher in abundance, if 4-methylbenzoate was additonally present. in the absence of benzoate, dctp and pdhb levels were unaffected by 4-methylbenzoate, agreeing with co-utilization of succinate and 4-methylbenzoate. however, if provided together, 4-methylbenzoate and benzoate had an additive negative effect on these two proteins, which could contribute to the observed decreased succinate depletion during phase with the ternary substrate mixture. it should be noted, however, that the observed differences might be related to using either succinate or 4-methylbenzoate as adaptation substrate in the compared experiments. the abundance change of dctp and pdhb was incongruent in the presence of succinate. i.e., dctp levels increased from − - to − -fold in phase while pdhb levels remained minimal at − -fold, thus indicating that unlike dctp, repression of pdhb is unaffected by the co-substrate succinate. discussion benzoate is preferred by magnetospirillum sp. strain pmbn1 magnetospirillum sp. strain pmbn is currently the only described isolate capable of anaerobically degrading 4-methylbenzoate. anaerobic degradation of 4-methylbenzoate and benzoate proceeds via two analogous pathways each requiring a distinct set of enzymes. despite adaptation to 4-methylbenzoate and thereby “transferred” initial presence of catabolic proteins for 4-methylbenzoate degradation, magnetospirillum sp. strain pmbn preferred utilization of benzoate from mixtures with 4-methylbenzoate. this preference cannot be attributed to markedly different growth rates. however, lower growth yields − for benzoate) despite the higher free energies obtained from complete oxidation of 4-methylbenzoate, indicate less efficient metabolism with 4-methylbenzoate. in addition, the range of substrates feeding into the 4-methylbenzoyl-coa degradation pathway is to our present knowledge limited to 4-methylbenzoate, whereas from the aromatic substrates of magnetospirillum sp. strain pmbn, at least are known from other denitrifying bacteria to be channeled into the central anaerobic benzoyl-coa pathway. indeed, also phenylacetate repressed 4-methylbenzoate utilization in magnetospirillum sp. strain pmbn. one may speculate that benzoate preference of magnetospirillum sp. strain pmbn reflects the higher probability to encounter substrates of the anaerobic benzoyl-coa pathway in the environment. in thauera aromatica ar- benzoate repressed anaerobic -dihydroxybenzoate degradation, which is apparently degraded via a catabolic route distinct from the anaerobic benzoyl-coa pathway. it seems thus likely that the anaerobic benzoyl-coa pathway might generally be preferred over more specialized aromatic degradation pathways. aerobic and anaerobic aromatic compound degrading bacteria in most reported cases prefer succinate over aromatic acids. the opposite was described recently for the first time in the denitrifying betaproteobacterium “a. aromaticum” ebn prefering benzoate despite adaptation to anaerobic growth with succinate. in this study, the same unusual preference was observed in the alphaproteobacterium magnetospirillum sp. strain pmbn which is phylogenetically distinct from “a. aromaticum” ebn in addition to succinate, growth behavior and substrate preferences of both strains were similar with mixtures of benzoate and the adaptation substrates fumarate or malate and acetate, but differed for oxaloacetate and pyruvate. while “a. aromaticum” ebn grew monophasically with one of the latter and benzoate, diauxic growth with benzoate preference was observed in magnetospirillum sp. strain pmbn substrate preferences in both strains could not be attributed to differences in growth rate and complexity of degradation pathways. taken together, magnetospirillum sp. strain pmbn and “a. aromaticum” ebn seem to be highly specialized on utilization of aromatic acids under anoxic conditions, since aliphatic substrates were first and exclusively utilized only from two tested binary substrate mixtures. benzoate mediates repression of 4-methylbenzoate uptake 4-methylbenzoate-specific detection of a putative abc transporter for 4-methylbenzoate uptake and the strong increase in transcript levels of its periplasmic solute-binding protein upon benzoate depletion, suggest benzoate to mediate repression of 4-methylbenzoate uptake. this is supported by non-utilization of 4-methylbenzoate during phase despite presence of some catabolic proteins. expression of genes for anaerobic catabolism of aromatic acids is typically controlled by regulatory proteins recognizing corresponding aryl-coa esters. inhibition of 4-methylbenzoate uptake would prevent its intracellular presence and thus generation of 4-methylbenzoyl-coa as putative inductor for expression of genes for 4-methylbenzoate catabolism. this is evident, e.g., from observed minimal transcript levels of mcla, encoding 4-methylbenzoate-coa ligase, and mbrc during phase it is known that benzoate-coa ligases of azoarcus sp. strain cib, t. aromatica k and magnetospirillum sp. strain ts- also convert 4-fluorobenzoate at high rates, and 3-methylbenzoate-coa ligase of azoarcus sp. strain cib exhibits the same activity with 3-methylbenzoate and benzoate. the high amino acid sequence identities of benzoate-coa ligase of magnetospirillum sp. strain pmbn and mbda of azoarcus sp. strain cib may suggest bcla of magnetospirillum sp. strain pmbn to possibly also accept 4-methylbenzoate as substrate. in this case, excluding 4-methylbenzoate from entering the cell would most effectively prevent induction of the 4-methylbenzoyl-coa pathway. benzoate mediates a dual repression of succinate uptake and utilization in case of “a. aromaticum” ebn it was assumed that benzoate mediates complete repression of succinate uptake by negatively controlling dctsr-dependent activation of dctpqm expression. despite the similar diauxic growth behavior and presence of homologous dctpqm/dctsr, magnetospirillum sp. strain pmbn partially depleted succinate already during benzoate utilization. this may be due to presence of an additional transporter for c4-dicarboxylate uptake together with a cognate c4-dicarboxylate-responsive two-component sensory/regulatory system in magnetospirillum sp. strain pmbn simultaneous employment of dcta and dctpqm in succinate uptake has been reported for pseudomonas aeruginosa pao where dctbd controls the coordinate expression of dcta and dctpqm. in magnetospirillum sp. strain pmbn succinate-specific detection of dcta and dctpqm indeed suggests participation of both transporters in succinate uptake. the coding genes are apparently coordinately expressed, considering the similar dynamics in their transcript levels across phases 1–. the only slightly reduced levels of c4-dicarboxylate transporter transcripts during phase suggest that benzoate does not have a similarly strong negative effect on dctsr- and/or dctbd-dependent transcriptional activation of dctpqm/dcta expression in magnetospirillum sp. strain pmbn compared to “a. aromaticum” ebn together with the partial depletion of succinate already during phase repression of succinate uptake cannot be the only determinant for the observed diauxie in magnetospirillum sp. strain pmbn succinate catabolism involves reactions of the tca cycle, as well as malic enzyme and pyruvate dehydrogenase to generate acetyl-coa. reduced transcript/protein levels of the latter two enzymes in magnetospirillum sp. strain pmbn during benzoate utilization in phase, suggest benzoate to also mediate inhibition of succinate conversion to acetyl-coa. the abundance decrease of the pdhb transcript, which was most pronounced and prolonged until complete benzoate depletion, indicates that benzoate could mediate transcriptional repression of pyruvate dehydrogenase as main target for inhibition of succinate catabolism. since expression of pyruvate dehydrogenase genes requires intracellular pyruvate as inductor in s. meliloti and e. coli, one may speculate that due to impeded succinate uptake together with reduced maeb-dependent pyruvate generation, intracellular pyruvate levels are insufficient for activation of pyruvate dehydrogenase gene expression. the partial depletion of pyruvate during preferred benzoate utilization could suggest a more direct involvement of benzoate in repressing the expression of pyruvate dehydrogenase genes. apparently, also here full expression of the pyruvate dehydrogenase genes as basis for maximal pyruvate consumption could only be achieved upon complete depletion of benzoate. uptake of succinate, l-malate and fumarate in magnetospirillum sp. strain pmbn most likely proceeds via dctpqm and dcta, whereas these transporters are generally not involved in import of pyruvate, oxaloacetate or acetate. with the exception of acetate, pyruvate dehydrogenase is essential for intracellular conversion of these five aliphatic acids to acetyl-coa. co-utilization of acetate from a mixture with benzoate thus agrees with the importance of pyruvate dehydrogenase in achieving preferential benzoate utilization in magnetospirillum sp. strain pmbn possible regulatory mechanisms for simultaneous repression of 4-methylbenzoate and succinate utilization at present we can only speculate about the mechanism underlying benzoate-mediated repression of 4-methylbenzoate and succinate utilization in magnetospirillum sp. strain pmbn in case of 4-methylbenzoate, a possible regulatory mechanism could involve the predicted tetr-type transcriptional regulator orf and benzoyl-coa as the common intermediate of anaerobic benzoate and phenylacetate degradation. one may speculate that in analogy to the tetr-type repressor dest, benzoyl-coa may act as co-repressor of orf re-establishing transcriptional repression of genes for 4-methylbenzoate uptake and catabolism in cells that were originally adapted to 4-methylbenzoate. on the contrary, shared intermediates occuring in the anaerobic degradation of benzoate, 4-methylbenzoate, acetate, phenylacetate or 4-hydroxybenzoate, suggest benzoate itself as inductor to mediate the dual repression of c4-dicarboxylate uptake and catabolism. one may speculate that benzoate interferes directly or indirectly with dctsr- and/or dctbd-mediated signal transduction, e.g. by modulating kinase activities. in addition, benzoate may activate transcriptional repressor, facilitate degradation of target transcripts, or negatively affect translation of transcripts at the sensory/regulatory, uptake and catabolic level.  next to “a. aromaticum” ebn the only distantly related magnetospirillum sp. strain pmbn is the second reported pure culture displaying an unexpected diauxie with benzoate preference from mixtures with c4-dicarboxylates. despite the similar growth behavior, apparently different molecular mechanisms govern the sequential utilization in both strains, adding to the so far largely unexplored regulatory potential within anaerobic aromatic compound-degrading bacteria. preferential utilization of aromatic acids, or their co-utilization with aliphatic acids, seems to be more common among these degradation specialists than previously expected. co-utilization even at the applied high concentrations of aromatic and aliphatic acids further suggests only weak or actually absent carbon catabolite control. the unique nutritional feature of magnetospirillum sp. strain pmbn is the capacity to anaerobically grow with 4-methylbenzoate, in addition to aromatic acids degraded via the widespread anaerobic benzoyl-coa pathway. one may speculate that the observed temporal separation of benzoate and 4-methylbenzoate utilization, and thereby successive operation of the benzoyl-coa and 4-methylbenzoyl-coa pathways allow preventing metabolic inefficiencies, e.g. by unbalanced draining of shared co-factors or the competitive inhibition of analogous enzymes. 
diauxic growth with benzoate preference was observed for binary and ternary substrate mixtures containing 4-methylbenzoate and succinate. apparently, simultaneous repression of 4-methylbenzoate and succinate utilization from the ternary substrate mixture resulted from inhibition of 4-methylbenzoate uptake, and combined inhibition of succinate uptake and succinate conversion to acetyl-coa. anaerobic degradation4-methylbenzoatearomatic compoundsrepressiondiauxieternary substrate mixturephysiologytranscript analysisproteomicsissue-copyright-statement© the author 2014 strain pmbn for benzoate from mixtures with 4-methylbenzoate, and thus temporal separation of the benzoyl-coa and 4-methylbenzoyl-coa pathway, may reflect a catabolic tuning towards metabolic efficiency and the markedly broader range of aromatic substrates feeding into the central anaerobic benzoyl-coa pathway. strain pmbn anaerobically degrades a wide variety of aliphatic and aromatic compounds and is unique for anaerobic degradation of 4-methylbenzoate. at high concentrations of organic substrates, microbial utilization of preferred substrates often results in diauxic growth with hierarchical substrate depletion. anaerobic growth and substrate consumption were monitored in binary and ternary mixtures of 4-methylbenzoate, benzoate and succinate, in conjuction with time-resolved abundance profiling of selected transcripts and/or proteins related to substrate uptake and catabolism. on the contrary, 4-methylbenzoate and succinate were utilized simultaneously from a binary mixture, as well as after benzoate depletion from the ternary mixture. considering the presence of these two different anaerobic “aromatic ring degrading” pathways, substrate preferences of magnetospirillum sp. the benzoate-mediated repression of c4-dicarboxylate utilization in magnetospirillum sp. unlike the carbon catabolite repression-mediated discriminative utilization of carbohydrates, the substrate preferences of non-carbohydrate-utilizing bacteria for environmentally relevant compound classes are rarely investigated. the preferential or simultaneous utilization of benzoate and other aromatic acids from mixtures with aliphatic acids may represent a more common nutritional behavior among degradation specialist than previously thought. the latter proceeds via a distinct reaction sequence analogous to the central anaerobic benzoyl-coa pathway to intermediates of central metabolism.
 at high concentrations of organic substrates, microbial utilization of preferred substrates often results in diauxic growth with hierarchical substrate depletion. unlike the carbon catabolite repression-mediated discriminative utilization of carbohydrates, the substrate preferences of non-carbohydrate-utilizing bacteria for environmentally relevant compound classes are rarely investigated. the denitrifying alphaproteobacterium magnetospirillum sp. strain pmbn anaerobically degrades a wide variety of aliphatic and aromatic compounds and is unique for anaerobic degradation of 4-methylbenzoate. the latter proceeds via a distinct reaction sequence analogous to the central anaerobic benzoyl-coa pathway to intermediates of central metabolism. considering the presence of these two different anaerobic “aromatic ring degrading” pathways, substrate preferences of magnetospirillum sp. strain pmbn were investigated. anaerobic growth and substrate consumption were monitored in binary and ternary mixtures of 4-methylbenzoate, benzoate and succinate, in conjuction with time-resolved abundance profiling of selected transcripts and/or proteins related to substrate uptake and catabolism.  diauxic growth with benzoate preference was observed for binary and ternary substrate mixtures containing 4-methylbenzoate and succinate. on the contrary, 4-methylbenzoate and succinate were utilized simultaneously from a binary mixture, as well as after benzoate depletion from the ternary mixture. apparently, simultaneous repression of 4-methylbenzoate and succinate utilization from the ternary substrate mixture resulted from inhibition of 4-methylbenzoate uptake, and combined inhibition of succinate uptake and succinate conversion to acetyl-coa. the benzoate-mediated repression of c4-dicarboxylate utilization in magnetospirillum sp. strain pmbn differs from that recently described for “aromatoleum aromaticum” ebn.  the preferential or simultaneous utilization of benzoate and other aromatic acids from mixtures with aliphatic acids may represent a more common nutritional behavior among degradation specialist than previously thought. preference of magnetospirillum sp. strain pmbn for benzoate from mixtures with 4-methylbenzoate, and thus temporal separation of the benzoyl-coa and 4-methylbenzoyl-coa pathway, may reflect a catabolic tuning towards metabolic efficiency and the markedly broader range of aromatic substrates feeding into the central anaerobic benzoyl-coa pathway. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords anaerobic degradation4-methylbenzoatearomatic compoundsrepressiondiauxieternary substrate mixturephysiologytranscript analysisproteomicsissue-copyright-statement© the author 2014 