Indian Journal of Biochemistry & BiophysicsVol. 42, February 2005, pp. 28-33

Effect of iron concentration on the expression and activity ofcatalase-peroxidases in mycobacteria

Veena C Yeruva, CAS Sivagami Sundaram and Manjula Sritharan*Department of Animal Sciences, School of Life Sciences, University of Hyderabad,

Hyderabad 500 046, India

Received 10 June 2004; revised 25 November 2004

Mycobacterial catalases are known to exist in different isoforms. We studied the influence of iron concentration on theexpression and activity of the different isoforms in Mycobacterium bovis BCG, M. smegmatis, M. fortuitum, M. kansasii andM. vaccae by growing them under iron-sufficient (4 ug Fe/mL) and iron-deficient (0.02 ug Fe/ml) conditions. Upon irondeprivation, significant differences were observed in the catalase/peroxidase activities in both quantitativespectrophotometric assays and in the activity staining in native gels. Notable feature was that the peroxidase activity showeda significant decrease upon iron deprivation in all the mycobacteria, "except M. vaccae. Peroxidase activity in all themycobacteria, irrespective of the iron status was susceptible to heat inactivation. However, the isoforms of catalase showeddifferences in their heat stability, indicating possible structural differences in these proteins. For example, M. bovis BCGexpressed a heat labile catalase under iron-sufficient conditions, while a heat stable catalase band of similar mobility wasexpressed under iron-deprivation conditions. The study clearly indicates that iron plays an important role in the regulation ofexpression of the different isoforms of the catalase-peroxidases,

Keywords: catalase-peroxidases, mycobacteria, iron.

IPC Code: C12R 1/32

Microorganisms though face conditions of irondeprivation, as Fe (III) exists either as insolubleFe(OH)3 at neutral pH or as protein-bound iron in themammalian system, held by transferrin, lactoferrinand ferritin, they adapt to such conditions byelaborating unique mechanisms of iron acquisition'.Mycobacteria also adapt to the iron-deficientcondition and express siderophores (intracellularmycobactin and extracellular exochelin) and iron-regulated envelope proteins (IREPs) ,,2. Interestingly,IREPs have also been demonstrated in the in vivoderived mycobacteria'. The co-ordinated expressionof the siderophores and IREPs was demonstrated inMycobacterium neoaurum4

• At molecular level, IdeRiron regulator protein controls. the expression of thecomponents of iron acquisition machinery". The iron

*Corresponding authorPhone: + 091-40-2301-1763; 23134700Fax: +091-40-23010120E-mail: mssl@uohyd.ernet.in;srimanju@yahoo.comAbbreviations: IREP, iron-regulated envelope protein; CAS,chrome azurol S; INH, isonicotinic acid hydrazide; TMBZ,tetrameth yIbenzidi ne

regulator Fur, first identified in gram-negativeorganisms is also found in mycobacteria. Two Fur-like proteins that are known to regulate several genesinvolved in iron metabolism have been identified inthe genome of M. tuberculosis6

• The JurA gene,coding for one of the two Fur-like proteins is locatedupstream of the katG gene, coding for catalase-peroxidase and the fur-katG organization is conservedin several mycobacteria'.

Catalases and peroxidases are oxido-reductasesinvolved in the molecular mechanisms of defenseagainst reactive oxygen species. Although theypossess striking similarities in the reactionmechanism, they have different residues involved inthe haeme cavity". The enzymes that exhibit eithercatalatic or peroxidative activity are common amonganimals, plants and microorganisms. Prokaryotesexpress catalase-peroxidases possessing both theactivities in a single protein molecule, suggesting thecommon phylogeny of the catalase and peroxidativeactivities during evolution. In mycobacteria, twoclasses of catalases, namely the T and Marerecognised. The T class is the heat labile enzyme with

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YERUVA et al.: MYCOBACTERIAL CATALASE-PEROXIDASE AND IRON

. peroxidase activity and the M class is the heat stableform with catalase activity". Multiple catalases havebeendemonstrated in mycobacteria'".

Earlier catalase",12 and iron concentrations'i':" havebeen implicated in the virulence of mycobacteria.Here, we have attempted to investigate the inter-relationship between these components and studiedthe effect of iron concentration on the peroxidase andcatalase activities in different mycobacteria.

Materials and MethodsMycobacterialstrains

Mycobacterium smegmatis mc2155, M. kansasii(ATCC 12478), M. bovis BCG Danish (kindly givenby Dr. Peter Small, Stanford University, USA),M. vaccae and M. Jortuitum (National Repository forMycobacterial Cultures at Central JALMA InstituteforLeprosy, Agra, India) were used in the study.

Growth of mycobacteria under iron-sufficient and iron-deficientconditions

The different mycobacterial strains were grown inmodified Proskauer and Beck medium' under iron-sufficient (4 ~g Fe/ml) and iron-deficient (0.02 ugFe/ml)conditions.

Assayfor the expression of siderophoresThe establishment of iron-deficient conditions was

assayed by the universal chrome azurol S (CAS)assay". Briefly, 1 mL of culture filtrate was added to2 mL of CAS solution and the absorbance at 630 nmwas measured after 30 min incubation at roomtemperature. The concentration of the siderophore isexpressed as siderophore units. One siderophore unit=(Ae-As/Ad x 100, where Ac represents theabsorbance of CAS solution plus medium and As, theabsorbance of the CAS solution plus culture filtrate ofthe respective sample.

Preparation of cell-free cell extractsThe cells, washed and re-suspended in phosphate

buffer (0.05 M, pH 7.0) were sonicated in a Vibra cellsonicator (12 pulses with each pulse for 20 see at12 Hz). The sonicate was centrifuged at 8000xg for20 min to obtain the cell-free extract, which wasstored at -20°e. Protein concentration was estimatedby the method of Lowry et al.'6

J

EnzymeassaysCatalase activity was determined spectrophoto-

metrically'? by measuring the decrease in H202concentration by reading the absorbance at 240 nm

1t

(E240= 0.0435 mM-' cm') and the specific activity ofcatalase was expressed as micro moles of H202decomposed per min per mg of total protein.Peroxidase activity was assayed in a microtitre plateby incubating with the tetramethyl benzidine (TMBZ)substrate in citrate buffer at pH 5.0 (ready-to-usesolution purchased from Bangalore Genei, India). Theactivity was expressed as 00450nm units per mg oftotal protein.

Activity staining for catalase in non-denaturing native gelsNative PAGE of the cell-free extracts was

performed under non-denaturing conditions. Thebifunctional catalase-peroxidase activity was detectedby the double staining method".

ResultsEstablishment of iron deficient conditions: CAS assay

Iron-deficiently grown (0.02 ug Fe/ml) M. kansasii,M. Jortuitum, M. smegmatis and M. bovis BCG

• expressed significant levels of the siderophore ascompared to iron-sufficient cells (4 ug Fe/ml) and inM. vaccae, no appreciable difference was noted(Fig. 1).

Catalase-peroxidase activities in iron-sufficient and iron-deficient mycobacteria

Iron deprivation resulted in a 3-5-fold decrease inthe catalase activity in M. Jortuitum and M. kansasiiand a 2-fold decrease in M. bovis BCG andM. smegmatis. Under iron-sufficient conditions,

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Fig. I-Universal CAS assay demonstrating the expression ofsiderophores in M. bovis BCG, M. fortuitum, M. kansas ii, M.smegmatis and M. vaccae [The extracellular siderophore wasassayed by measuring the difference in the absorption at 630 nmas described in the 'Materials and Methods': Values are expresedas siderophore units. The vertical bars represent the standard de-viation of the mean from 3 independent experiments]

29

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30 INDIAN J. BIOCHEM. BIOPHYS., VOL. 42, FEBRUARY 2005

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Fig. 2-Spectrophotometric assay of catalase (A) and peroxidase(B) in iron-sufficient and iron-deficient cells of M. bovis BCG. M.

Jortuitum, M. kansas ii, M. smegmatis and M. vaccae [The catalaseactivity was measured by the decrease in the H202 concentrationand the peroxidase by reactivity with the TMBZ substrate as de-scribed in 'Materials and Methods'. The vertical bars represent thestandard deviation of the mean from 3 independent experiments]

M. kansasii expressed highest level of catalase activityand M. smegma tis the least. The notable exception wasM. vaccae which showed no significant change in thecatalase activity upon iron deprivation (Fig. 2A).

Peroxidase activity of M. bovis BCG, M. Jortuitumand M. kansasii was significantly decreased (10-13-fold) upon iron deprivation, while a smaller decreasewas observed in M. smegmatis. M. vaccae showed nochange in the activity with reference to the ironconcentration in the culture medium (Fig. 2B). Evenunder iron-sufficient conditions, the peroxidaseactivity of M. smegma tis and M. vaccae was muchlower than other mycobacteria.

Activity staining of catalase-peroxidase in native gelsThe effect of iron concentration on the expression

of the different isoforms of catalase-peroxidase andtheir susceptibility to heat treatment was visualized in

c2 3 4

D2 " 3 4

Fig. 3--Activity staining of native gels for catalase and peroxi-dase (method of Wayne and Diaz8). [Lanes 1 and 2, the enzymeactivities of iron-sufficient and iron-deficient M. bovis BCG (A),M. smegmatis (B), M. vaccae (C) and M. Jortuitum (D); and lanes3 and 4, the activity of identical extracts subjected to heat treat-ment at 55°C for 10 min]

native gels by activity staining (Fig. 3). The differentbands reflect only the mobility of the enzyme in thegel and do not indicate whether the enzymes withsimilar gel mobility are identical. Hence, the bandsare considered as distinct proteins. The differentisoforms of the catalase-peroxidase show variation,with bands of either catalase or peroxidase activity orboth as seen in M. bovis BCG (Fig. 3A), M.smegmatis (Fig. 3B), M. vaccae (Fig. 3C) M.Jortuitum (Fig. 3D) and M. kansasii (Fig. 4). Thedifferences in the enzyme activities between iron-sufficient and iron-deficient cells of the sameorganism and their susceptibility to heat treatment arenoteworthy. The observations in the differentmycobacteria are summarized in Table 1.

The progressive inactivation of the peroxidase andcatalase activities with increasing temperature wasseen in all mycobacteria tested and the observations inM. kansasii are presented. The quantitative decreasein the peroxidase (Fig. 4A) and catalase activities

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YERUV A et al.: MYCOBACTERIAL CATALASE-PEROXIDASE AND IRON 31

Table l---{:haracteristics of the multiple catalase-peroxidases of mycobacteria grown under iron-sufficient (4 Ilg Fe/ml) andiron-deficient (0.02 ug Fe/ml) conditions

[Thedifferent isoforms of mycobacterial catalases and peroxidases were separated by native PAGE and detected by the double stainingmethodas described in the 'Materials and Methods'. Differences in the enzyme activities and their heat stability were observed between

iron-sufficient (4 ug Fe/ml) and iron-deficient cells (0.02 ug Fe/ml)]

Species Iron status* Isoforms Enzyme activity Characteristics of catalase/peroxidase

t 2 Dual Significant peroxidase activity in both isoforms. Both peroxidase and catalaseM. bovisBCG t heat labile

2 Catalase Complete loss of peroxidase; one significant heat stable catalase

t 4 Dual-l Low level of peroxidase; both peroxidase and catalase are heat labilePeroxidase-2 Significant peroxidase; heat labile

M.[ortuitum Catalase-I Significant catalase; heat labile

t 3 Peroxidase-2 Low levels of activity; heat labileCatalase-I heat labile

t 4 Dual-3 Significantly high peroxidase and catalase activity; both were heat labile

M. kansasii Catalase-l Complete loss of peroxidase; one significant heat stable catalase• 2 Catalase-2 One major band of heat stable catalase activity. Another band showed weak .activity.

t 4 Catalase-l Heat stable catalase

M. vaccae Dual-3 Heat labile peroxidase, with one band of heat resistant catalase

+ 3 Dual-2 heat labile peroxidase and catalaseCatalase-I Prominent band of heat stable catalase

t 4 Catalase-I Heat stable catalase

M. smegmatis Peroxidase-3 One prominent band, with two faint bands. All were heat labile.

+ 1 Dual-l Significant catalase with weak peroxidase activity, both of which wereunaffected by heat

*Thearrows" and T indicate iron-sufficient and iron-deficient conditions of growth

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Fig. 4-Effect of increasing temperature on the catalase and peroxidase activities of iron-sufficient and iron-deficient cells of M. kansasii[The peroxidase (A) and catalase (B) activities were assayed both spectrophotometrically as described in 'Materials and Methods']

32 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 42, FEBRUARY 2005

(C)

2 3 64 7 8 9 10

Fig. 4C-Activity staining of native gels showing the effect oftemp. on catalase and peroxidase activities of iron-sufficient andiron-deficient cells of M. kansasii [The lanes I, 3, 5, 7 and 9represent the cell-free extracts of iron-sufficient cells and lanes2, 4, 6, 8 and 10 represent iron-deficient cells incubated for 30min at temp. CC) of 37,40,45,50 and 60 respectively]

(Fig. 4B) with increasing temperature were alsoreflected in the activity staining of identical samples(Fig. 4C). The differences in the susceptibilities ofdifferent isoforms to heat were evident from theactivity staining.

DiscussionIn this study, conditions of iron deprivation were

successfully established for M. [ortuitum, M. kansasii,M. smegmatis and M. vaccae and for the slowgrowing M. bovis BCG, as assessed by the CASassay. The most notable observation was the sharpdecrease in the peroxidase activity upon irondeprivation; the effect was highly pronounced in M.kansas ii. M. Jortuitum and M. bovis BCG. Thedistribution of high levels of peroxidase activityamong the various isoforms in iron-sufficient cellswas variable in the different mycobacteria. Theperoxidase activity, irrespective of the iron status washeat labile in the mycobacteria tested. In M. vaccae,though no significant differences in the catalase andperoxidase activities were observed spectrophoto-metrically between iron-sufficient and iron-deficientcells, qualitative differences could be seen uponactivity staining.

Interestingly, under conditions of iron deprivation,the catalase activity was not affected to a great extent.Contrary to the general notion that all catalases areheat stable'", we have observed that the catalasesexpressed by iron-deficient cells were more heatstable, as compared to the iron-sufficient cells. Forexample, in M. bovis BCG, the catalase expressed byiron-sufficient cells was heat labile, while under iron-deficient conditions, the catalase band showing

similar mobility was heat stable. It is likely thatstructural differences contribute to their susceptibilityto heat. Whether this alteration in the enzymeactivities confers an advantage to the organism inenvironments of varying iron concentration needs tobe studied further.

Thus, it can be inferred from the aboveobservations that in mycobacteria used in the study,the expression of different isoforms of the catalase-peroxidase with alterations in the enzyme activities isregulated at the molecular level by iron. It is reportedearlier that the FurA protein controls the expression ofthe katG gene:". Recently, it has been shown that theregulation of the katG gene by the Fur A protein ismuch more complex.", as two more regions pJurA andpkatG were identified in the control of expression ofthe katti gene. Although the exact mode of the FurAaction i§ not yet clear, the control of catalase-peroxidase expression at the post-transcriptional levelby the FurA was considered as likello. It is now clearin many bacteria that iron acts as a regulator"; uponbinding to Fur and Fur-like repressor, it controls theexpression of both the components of the iron-acquisition machinery and bacterial toxins. Thepresent study shows the direct effect of ironconcentration on the catalase and peroxidase activitiesin mycobacteria, the implications of which need to bestudied further.

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AcknowledgementThe financial assistance for establishment of the P3

facility and fellowship to one of us (VCY) by the AP-Netherlands Biotechnology Programme is gratefullyacknowledged. CASS is the recipient of thefellowship from CSIR, India. M S acknowledges theSpecial Assistance Program of the UGC, India forfinancial support to the Department for AnimalSciences, University of Hyderabad.

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.vitiesto be

he P3~AP-efully

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