the -subdivision of proteobacteria includes gram - negative bacteria with diverse and contrasting lifestyles , ranging from free - living , symbiotic to pathogenic bacteria . despite this apparent heterogeneity ,
-proteobacteria share some unexpected common features such as an asymmetric cell division followed by a differentiation process .
as the large nad - dependent glutamate dehydrogenases , to which gdhz belongs , are particularly well conserved among -proteobacteria , we postulated that the cell division control found in c. crescentus might be conserved as well . to address this question we created an in - frame deletion of gdhz homolog in the facultative intracellular pathogen brucella abortus 2308 ( gdhzba ) . similarly
to gdhz loss - of - function mutants in c. crescentus , the  gdhzba strain had a strong growth defect when grown in complex medium ( figure  2a ) .
interestingly , gdhzba cells were slightly elongated and branched ( figure  2b ) , which typically indicates a cell division defect in bacteria growing from one pole .
furthermore growth and cell division defects were suppressed when glucose or xylose was added to the complex media or when xylose was used as the sole carbon source in a synthetic medium ( figure  2a and data not shown ) .
altogether these observations suggest that the role of gdhz in controlling cell division might be conserved in b. abortus , and likely in other -proteobacteria . 

( a ) growth of b. abortus wild - type ( black ) and gdhzba ( gray ) cells in complex medium ( 2yt ) or in synthetic medium with xylose as the only carbon source ( plommet xylose ) , showing gdhz is required for optimal growth in complex mediium .
( b ) phase contrast imaging of b. abortus wild - type and gdhzba cells grown in complex medium ( 2yt ) illustrating the morphological defects with elongated and branched cells ( arrows ) developed by gdhzba cells .
( a ) growth of b. abortus wild - type ( black ) and gdhzba ( gray ) cells in complex medium ( 2yt ) or in synthetic medium with xylose as the only carbon source ( plommet xylose ) , showing gdhz is required for optimal growth in complex mediium .
( b ) phase contrast imaging of b. abortus wild - type and gdhzba cells grown in complex medium ( 2yt ) illustrating the morphological defects with elongated and branched cells ( arrows ) developed by gdhzba cells .
as an intracellular pathogen , brucellae reside preferentially within trophoblasts and macrophages , in which it can survive and proliferate to produce chronic infections .
intracellular brucellae hijack the intracellular vesicular trafficking to finally reach and replicate into the endoplasmic reticulum .
althougth widely accepted to be a nutrient poor environment , the exact composition of the brucella containing vacuole ( bcv ) remains unclear . to test whether gdhz is important for intracellular replication , murine raw macrophages were infected with either wild - type or gdhzba strain , and the number of intracellular bacteria was determined at 2 hrs , 24 hrs and 48  hrs post - infection ( pi ) .
as gdhz constitutes a key entry point into the tricarboxylic acid ( tca ) cycle for several amino acids , the gdhzba mutant would be unable to efficiently replicate inside host cells if amino acids were a main carbon source during infection .
as illustrated in figure  3a , the gdhzba mutant was already impaired 2 hrs pi and intracellular brucellae failed to replicate efficiently at 24 hrs and 48 hrs pi . during the first 68 hrs following entry into the cell , intracellular brucellae
therefore , the difference in the number of intracellular bacteria between the strains at 2 hrs post - infection might be due to a defect in the internalization process .
since bacteria were cultivated in complex media prior to infect , the proper entry of gdhzba cells inside macrophages might be affected by their morphological defect ( figure  2 ) . to circumvent this problem ,
b. abortus wild - type and gdhzba strains were grown in complex medium supplemented with glucose prior to infect murine macrophages .
interestingly , when glucose was added to the preculture medium , both wild - type and gdhzba intracellular bacteria were present at similar levels at 2 hrs pi , indicating that the entry impairment observed with gdhzba cells was very likely a secondary effect ( figure  3b ) . nevertheless , at 24 hrs and 48  hours pi , gdhzba cells failed to efficiently replicate compared to wild - type cells , whatever the medium used for cultivating brucella before infecting macrophages .
altogether these results strongly suggest that gdhz is required for optimal intracellular replication of brucella , and emphasize the importance of amino acids as a main carbon source for brucellae inside host cells . 

internalization and intracellular replication of b. abortus wild - type and gdhzba cells into murine raw macrophages .
raw 264.7 macrophages were cultured at 37 c ( 5 % co2 atmosphere ) in dmem ( invitrogen ) supplemented with 10% fetal bovine serum ( gibco ) , 4.5  g / l glucose , 1.5  g / l nahco3 and 4  mm glutamine .
bacteria were centrifuged at 400 x g for 10  min at 4 c and then incubated for 1 hr at 37 c ( 5 % co2 atmosphere ) .
cells were washed twice with fresh medium and then incubated in medium supplemented with 50  g / ml gentamicin to kill extracellular bacteria .
prior to infections , bacteria were grown in ( a ) 2yt or ( b ) 2yt + 0.2 % glucose .
the significant pairwise comparisons are indicated for p < 0.5 ( * ) , p < 0.01 ( * * ) and p < 0.001 ( * * * ) in student t - tests . 
 
internalization and intracellular replication of b. abortus wild - type and gdhzba cells into murine raw macrophages .
raw 264.7 macrophages were cultured at 37 c ( 5 % co2 atmosphere ) in dmem ( invitrogen ) supplemented with 10% fetal bovine serum ( gibco ) , 4.5  g / l glucose , 1.5  g / l nahco3 and 4  mm glutamine
. cultures of brucella were prepared in dmem at a multiplicity of infection of 50 .
bacteria were centrifuged at 400 x g for 10  min at 4 c and then incubated for 1 hr at 37 c ( 5 % co2 atmosphere ) .
cells were washed twice with fresh medium and then incubated in medium supplemented with 50  g / ml gentamicin to kill extracellular bacteria .
prior to infections , bacteria were grown in ( a ) 2yt or ( b ) 2yt + 0.2 % glucose .
the significant pairwise comparisons are indicated for p < 0.5 ( * ) , p < 0.01 ( * * ) and p < 0.001 ( * * * ) in student t - tests .
other metabolic regulators of cell division have been described in escherichia coli and bacillus subtilis .
interestingly , all these regulators link glycolysis to cell division and both species use glucose and hexoses as major carbon sources . on the other hand , the deletion of gdha in e. coli , coding for an anabolic nadp - dependent glutamate dehydrogenase , did neither slow down growth in complex ( lb ) or synthetic ( m9 g ) media , nor interfere with cell division ( data not shown ) .
in contrast , c. crescentus and b. abortus use gdhz , catabolizing amino acids , to coordinate metabolism with cell division . based on these observations we postulate that bacteria have evolved by connecting the cell division machinery with the catabolic routes whose activity reflects nutrient fluctuations in the environment .
in other words , such regulations would underline the main carbon sources used by bacteria , indicating that amino acids would constitute a favorite carbon source for c. crescentus , b. abortus , and likely other -proteobacteria .
this hypothesis is further supported by the fact that amino acids transporters and catabolic enzymes are induced in bcv .
it is noteworthy that , whatever the main carbon source is , metabolic enzymes constitute perfect candidates to fulfill additional regulatory functions .
indeed , the conformational change induced upon substrate(s ) binding or product(s ) release , can serve as a proxy for the cell to monitor nutrient availability .
this is thereby not surprising that most of the proteins in the glycolytic and the tca pathways are moonlighting enzymes , i.e. enzymes with secondary ( regulatory ) functions .
in this perspective and knowing that a nad - dependent glutamate dehydrogenase connects the nitrogen cycle to the tca cycle , gdhz was very likely selected to coordinate growth with cell division in -proteobacteria .
f.b . held a fria ( fund for research training in industry and agriculture ) fellowship from the national fund for scientific research ( f.r.s .- fnrs ) .
this work was supported by a research project ( pdr t.0053.13 ) to x.d.b and a research credit ( cdr 1.5067.12 ) to r.h from the f.r.s .- fnrs .
 abstractprior to initiate energy - consuming processes , such as dna replication or cell division , cells need to evaluate their metabolic status . 
 we have recently identified and characterized a new connection between metabolism and cell division in the -proteobacterium caulobacter crescentus . 
 we showed that an nad - dependent glutamate dehydrogenase ( gdhz ) coordinates growth with cell division according to its enzymatic activity . here 
 we report the conserved role of gdhz in controlling cell division in another -proteobacterium , the facultative intracellular pathogen brucella abortus . 
 we also discuss the importance of amino acids as a main carbon source for -proteobacteria . 
GdhZ coordinates growth with cell division in the intracellular pathogen 
GdhZ is required for efficient intracellular replication of 
Amino acids as a main carbon source for -proteobacteria
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