Essential Genes actA

Mutations in this gene result in an inability to grow at pH 6 or to maintain intracellular pH (pH;) if the external pH (pHe) is less than 6.5 (O'Hara et al. 1989). The mutant (TG2-6) is also abnormally copper- and zinc-sensitive at pH 7.0, but removal of copper from the medium does not restore acid-sensitivity. Complementation resulting in restoration of acid tolerance also restores copper- and zinc-tolerance (Tiwari et al. 1996a). The deduced ActA protein sequence is suggestive of a membrane protein; its closest similarity is to the CutE protein of E. coli, damage to which also results in Cu-sensitivity (Rogers et al. 1991). In S. meliloti, actA homologs occur in all strains tested, and expression in WSM419 is constitutive (Tiwari et al. 1996a). The mutant in actA is rescued by calcium.

5. Essential Genes - actR and actS

In S. meliloti, these two contiguous genes form a sensor-regulator pair, mutation of either of which leads to acid-, zinc- and cadmium-sensitivity (Tiwari et al. 1996b); acid sensitivity also occurs with R. leguminosarum (Boesten et al. 2000). Expression of promoterless lacZ fused to either actS or actR is constitutive (Tiwari et al. 1996b), consistent with a system necessary for a major cellular stress response. A multi-copy plasmid (pRT546-6) carrying actS restores acid and metal tolerance to an acts' mutant but not to an actR' mutant. However, a multi-copy plasmid carrying actR restores tolerance to both types of mutant, possibly implying "cross-talk" to other sensor(s) or sufficient phosphorylated ActR for normal function. Calcium allows mutants with lesions in actS or actR to recover acid-tolerance. The adaptive acid tolerance response (ATR) is abolished by mutation of either actS or actR.

The deduced ActS sequence shows five strongly hydrophobic domains likely to be membrane-located, and a typical sensor C-terminal domain (Tiwari et al. 1996b). The deduced ActR sequence has an H-T-H motif NVSETARRLNMNRRTLQRILAK suggestive of a DNA-binding role; it has aspartate and lysine residues typical of regulators.

The sequences of the ActS and ActR proteins show marked similarities to a major group of global regulator systems, particularly those involved in regulation of photosynthetic systems (Eraso, Kaplan 1994, 1995; Joshi, Tabita 1996; Bauer, Bird 1996; Masuda et al. 1999) and of N2 fixation (RegSR) in Bradyrhizobium japonicum (Bauer et al. 1998; Fischer et al. 2000). However, in the N2-fixing S. meliloti WSM419, mutations in actS or actR do not result in a non-Ni-fixing nodule phenotype as they do in B. japonicum, implying a marked difference in function despite the great overall similarity to the RegSR system.

5.1. Genes responsive to ActS-ActR. To address the question of which genes are controlled by ActS-ActR, we created random mutations in S. meliloti with a minitransposon carrying a protomoterless gusA (mTn5-GNm; Reeve et al. 1999) in a strain (RT295S) which was chromosomally acts' but complemented with a plasmid-borne actS, selecting for clones which expressed gusA differentially between pH 7.0 and 5.7. We then cured pH-selected mutants of the aciS-carrying plasmid by introducing an incompatible plasmid, and then inserted plasmids carrying either actR alone or actR (Fenner et al. 2000) and actS. These strains were then examined to compare expression of GusA in broth cultures against that for strains carrying only the vector plasmid, the results leading to identification of a number of genes whose expression is modulated by the ActS-ActR system.

The genes so identified include two hypothetical S. meliloti proteins, ribulose-fc-phosphate carboxylase (small subunit), a nitrate reductase component, and fixN202 (ebb3 cytochrome oxidase components) (Table 1), though only the last has any obvious connection to a possible mechanism for an acid protection mechanism.

6. Essential Genes - actP

Mutants with lesions in actP in S. meliloti (RT3-27) or R. leguminosarum bv. viciae (WR1-14) are acid-sensitive but not rescuable with calcium. They are both also specifically copper-sensitive (no change in sensitivity to cadmium, mercury, silver or zinc) compared to the wild type; removal of copper from the minimal medium also restores acid tolerance. The two species are markedly different in their copper tolerance. In a minimal medium at pH 5.7, S. meliloti WSM419 tolerates only 50 |_iM

added C11SO4 whereas R. leguminosarum bv viciae WSM710 tolerates 500 fxM. This sensitivity is also markedly influenced by the medium; with glutamate as the N-source much higher concentrations of copper are tolerated than when NH4CI is used, because of chelation of copper by glutamate.

Table 1. actS-actR-responsive gusA fusions i n S. meliloti.

Mutant

Gene

Putative function

Location

RTA6S

ORF4059

Hypothetical protein

Chromosome

RTA15S

cbbS

Ribulose-fc-phosphate carboxylase small subunit

pSymB

RTG47S

narB

Assimilatory nitrate reductase large subunit

pSymB

RTH48S

ORF2871

Binding-protein dependent transporter

Chromosome

RTL19S

ORF795

Hypothetical protein

Chromosome

RTM11S

hyuA

Hydantoin utilization protein

pSymB

RTN37S

gstl

Glutathione S-transferase

Chromosome

RT033S

ORF888

Two component receiver protein

Chromosome

BF1212S

fixN202

ebbs cytochrome oxidase components

pSymA

The sequences of the inactivated genes are very similar and typical of P-type ATPases. They belong to the CPx sub-family of the ATPases that recognize and transport heavy metals. In R. leguminosarumWSMHO, expression of an actP-gusA fusion is induced specifically by increasing copper concentrations at both pH 5.7 and 7.0. For the same concentration of added copper the induction is much greater at pH 5.7 than at 7.0, probably because decreased chelation at the lower pH results in a much greater copper concentration.

Downstream from actP in both organisms is hmrR, the sequence of which places it in the merR family of heavy metal regulators. Because of the copper induction of actP, we made sure that the inactivation of actP with Tn5 was not a polar effect on hmrR by showing complementation of the actP mutation with a DNA segment carrying actP with an active hmrR, or with one where the hmrR had been insertionally inactivated. When the chromosomal copy of hmrR is inactivated in WSM710, the basal level of expression of an actP-gusA fusion at both pH 7.0 and 5.7 is increased, but the response to copper concentration is abolished. HmrR therefore appears to be a second member of the MerR family responsive to copper and to belong to the CopR subgroup (Peterson, Moller 2000); unlike the copR-copA system in E. coli, HmrR appears to be both a positive and negative regulator.

7. Essential Genes - exoH

S. meliloti WRR1 is a Tn5-induced mutant of WSM419 isolated as being highly acid-sensitive; it also has a zinc-sensitive phenotype. As with the exoH mutant from S. meliloti 1021 (Leigh et al. 1987), its EPS1 is not succinylated, nor does the high molecular weight form normally get cleaved to low molecular weight forms. However, the symbiotic phenotype in WRR1 is not one of empty ineffective nodules as in strain 1021. Sub-cloning from an 18 kb fragment carrying exol to exoP and contiguous genes from 1021 (Leigh et al. 1987) indicates that a fragment containing only exoH complements both acid- and zinc-sensitive phenotypes. The mechanism behind the acid- and zinc-sensitivity of WRR1 is still obscure, but may involve succinylation of a cellular component other than EPS1.

What is remarkable about these acid-sensitive mutants is that all show additional metal-sensitive phenotypes (Table 2). This connection is unclear except in the case of actP, where copper-

associated induction of actP mediated by HmrR presumably allows the cell to export the increased amount of copper entering it as a result of low pH-induced increase in copper availability.

Table 2. Metal sensitivities of acid-sensitive mutants.

Organism

Mutant

Lesion in

Rescue by

Sensitivity response to

Ca2+

Low pH

Cu2+

Cd2+

Zn2+

S. meliloti

TG2-6

actA

Yes

Yes

Yes

Yes

S. meliloti

TG5-46

actR

Yes

Yes

Yes

Yes

S. meliloti

RT295

actS

Yes

Yes

Yes

Yes

S. meliloti

RT3-27

actP

No

Yes

Yes

R. leguminosarum

WR1-14

actP

No

Yes

Yes

S. meliloti

WRR1

exoH

Yes

Yes

Yes

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