Characterization of Xanthomonas oryzae pv. oryzae recX, a gene that is required for high-level expression of recA.

Analysis of the nucleotide sequence downstream from the Xanthomonas oryzae pv. oryzae recA gene reveals two orfs designated orfX and recX. The former has the potential to code for a 5.6 kDa protein of unknown function while the latter encodes for a putative 14.6 kDa protein with homology to RecX from various bacteria. Northern blot analysis and RT-PCR results show that recA-orfX-recX are co-regulated and arranged in an operon. A recX mutant was constructed. The mutant has no obvious growth defects or stress response defects, except that it cannot support high-level expression of recA from an expression vector. Introduction of the plasmid containing recA into the recX mutant resulted in reduced transformation efficiency and all transformants tested had mutations with reduced RecA levels. Moreover, the recX mutant has reduced basal levels of RecA. This has not been observed in other bacteria. When inactivated recX was complemented in trans, both changes were reversed. recX mutation has no effect on the regulation of the recA promoter, suggesting that its effect on the RecA level could be post-transcriptional.

Complex regulation of the organic hydroperoxide resistance gene (ohr) from Xanthomonas involves OhrR, a novel organic peroxide-inducible negative regulator, and posttranscriptional modifications.

Analysis of the sequence immediate upstream of ohr revealed an open reading frame, designated ohrR, with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression. ohrR was transcribed as bicistronic mRNA with ohr, while ohr mRNA was found to be 95% monocistronic and 5% bicistronic with ohrR. Expression of both genes was induced by tert-butyl hydroperoxide (tBOOH) treatment. High-level expression of ohrR negatively regulated ohr expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the ohrR promoter (P1) has organic peroxide-inducible, strong activity, while the ohr promoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR. ohr primer extension results revealed three major primer extension products corresponding to the 5' ends of ohr mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical Xanthomonas promoter. Instead, the regions can form a stem-loop secondary structure with the 5' ends of ohr mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the ohrR-ohr operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of ohr mRNA, while ohrR mRNA is rapidly degraded.

Xanthomonas oryzae pv. oryzae recA is transcribed and regulated from multiple promoters.

Transcription regulation of Xanthomonas oryzae pv. oryzae recA was characterized. Primer extension experiments showed that recA is transcribed from three promoters designated P1, P2 and P3. The sequences of -10 and -35 regions of these promoters have moderate homology to the proposed consensus sequence for a Xanthomonas promoter. Putative SOS boxes were identified in the vicinity of P1 and P2 promoters. Deletion analysis and in vivo monitoring of promoter activity of these promoters revealed that the three promoters have different characteristics. P1 and P2 show stress-inducible high and low promoter strengths respectively. P3 is a non-inducible moderate promoter strength. These promoters are regulated by two SOS boxes. The multiplicity of promoters and SOS boxes provides back-up systems to ensure proper regulation of recA.

Construction and characterization of regulated L-arabinose-inducible broad host range expression vectors in Xanthomonas.

Several versions of broad host range (BHR), L-arabinose-inducible expression vectors were constructed. These expression vectors were based on a high copy number BHR pBBR1MCS-4 replicon that could replicate in both enteric and non-enteric Gram-negative bacteria. Two versions of expression cassettes containing multiple cloning sites either with or without a ribosome binding site were placed under transcriptional control of the Escherichia coli BAD promoter and araC gene. Three versions of vectors containing ampicillin or kanamycin or tetracycline resistance genes as selectable markers were constructed. In all six new L-arabinose-inducible BHR expression vectors containing many unique cloning sites, selectable markers were made to facilitate cloning and expression of genes in various Gram-negative bacteria. A Tn9 chloramphenicol acetyl transferase (cat) gene was cloned into an expression vector, resulting in pBBad18Acat that was used to establish optimal expression conditions (addition of 0.02% L-arabinose to mid-exponential phase cells for at least 1 h) in a Xanthomonas campestris pv. phaseoli. Comparison of the Cat enzyme activities between uninduced and a 180-min L-arabinose-induced culture showed a greater than 150-fold increased Cat specific activity. In addition, L-arabinose induction of exponential phase cells harboring pBBad18Acat gave a higher amount of Cat than similarly treated stationary phase cells. The usefulness of the expression vector was also demonstrated in both enteric and non-enteric Gram-negative bacteria.

Construction and physiological analysis of a Xanthomonas mutant to examine the role of the oxyR gene in oxidant-induced protection against peroxide killing.

We constructed and characterized a Xanthomonas campestris pv. phaseoli oxyR mutant. The mutant was hypersensitive to H2O2 and menadione killing and had reduced aerobic plating efficiency. The oxidants' induction of the catalase and ahpC genes was also abolished in the mutant. Analysis of the adaptive responses showed that hydrogen peroxide-induced protection against hydrogen peroxide was lost, while menadione-induced protection against hydrogen peroxide was retained in the oxyR mutant. These results show that X. campestris pv. phaseoli oxyR is essential to peroxide adaptation and revealed the existence of a novel superoxide-inducible peroxide protection system that is independent of OxyR.

Characterization and expression analysis of a Xanthomonas oryzae pv. oryzae recA.

Nucleotide sequence of Xanthomonas oryzae pv. oryzae (Xoo) DNA from pSM-A1 was determined and sequence analysis revealed an ORF with high homology to RecA proteins. Expression analysis using an anti-RecA antibody demonstrates that MMS treatment induces recA in Xanthomonas strains but not in an Escherichia coli harbouring cloned Xoo recA. This indicates the existence of a recA regulatory mechanism in Xanthomonas that is not function in E. coli. In Xoo, recA was highly induced by treatments with chemical mutagens, UV and peroxides, while superoxides, a thiol agent, a heavy metal and heat shock were not inducers. The increased amount of RecA induced by H2O2 or MMS treatments were due to increased transcription of recA. recA showed no growth phase or starvation regulation. The pattern of recA regulation in Xoo could play important roles in stress survival in the environment and during plant-microbe interactions.

Analysis of RNA polymerase gene mutation in three isolates of rifampicin resistant Mycobacterium tuberculosis.

Drug resistance in tuberculosis (TB) has become a major public health threat, particularly when the disease cannot be 100% controlled by BCG vaccination. In Thailand, resistance to rifampicin, a major component of multidrug regimens of treatment, is the common cause of tuberculosis recurrence. The mechanism of rifampicin resistance involves alterations of the RNA polymerase subunit beta (rpo B) gene. Mutations in rpo B gene were often found to cluster within a region of 23 amino acids starting from amino acid residue 511 to residue 533. Direct PCR sequencing was utilized to compare base changes in rpo B gene in three rifampicin resistant phenotypes of M. tuberculosis isolated from Thai patients. The sequences showed one base substitution at codon 531 resulting in an amino acid change from serine (TCG) to leucine (TTG) in a multidrug resistant isolate compared to that of a sensitive isolate, whereas a point mutation at codon 516 causing a change from aspartic acid (GAC) to tyrosine (TAC) was detected in a multidrug resistant isolate from a HIV positive patient. In an isolate resistant only to rifampicin a double mutation at codon 531 changing serine (TCG) to phenylalanine (TTT) was found. No mutations were observed in the same region in streptomycin, ethambutol or isoniazid resistant isolates. This finding reports two new types of mutation (GAC to TAC at codon 516 and TCG to TTT at codon 531) and confirms a direct correlation between rpo B gene alteration and rifampicin resistant phenotype in M. tuberculosis.